UNIVERSITÉ DU QUÉBEC À RIMOUSKI RÉSISTANCE...

UNIVERSITÉ DU QUÉBEC À RIMOUSKI

RÉSISTANCE DES OMBLES (Salvelinusfontinalis et Salvelinus alpinus)

AUX INFECTIONS BACTÉRIENNES OPPORTUNISTES EN MILIEU MARIN

MÉMOIRE

PRÉSENTÉ À

L'UNIVERSITÉ DU QUÉBEC À RIMOUSKI

comme exigence partielle

du programme de maîtrise en océanographie

PAR

ANNABELLE LEFRANT

Septembre 2006

UNIVERSITÉ DU QUÉBEC À RIMOUSKI Service de la bibliothèque

Avertissement

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REMERCIEMENTS

Je souhaiterais remercier toutes les personnes qui ont participé à la réalisation de ce projet,

et plus particulièrement ma directrice, le Dr Céline Audet de l'Institut des sciences de la

mer de Rimouski pour sa disponibilité, son dynamisme, ses nombreux encouragements et

son optimisme à toute épreuve! Merci également à mes deux co-directeurs, le Dr Pierre

Belhumeur de l'Université de Montréal, pour son accueil chaleureux au laboratoire, ainsi

que le Dr Jean-Marie Sévigny de l'Institut Maurice Lamontagne, pour la pertinence de ses

commentaires tout au long du projet. Je tiens aussi à remercier le Dr Karine Lemarchand de

l' Institut des sciences de la mer de Rimouski, pour sa collaboration au projet concernant le

volet bactériologique. Ses explications et sa disponibilité m'ont beaucoup aidé lors de la

réalisation du projet. Un gros merci à Sébastien Croisetière, agent de recherche à

l'Université de Montréal, Éric Parent et Geneviève Canuel, respectivement agent de

recherche et stagiaire à l'Institut Maurice-Lamontagne, pour leur aide très précieuse. Merci

également à Nathalie Morin et Dominique Lavallée pour leur assistance technique lors de

l' élevage des poissons à la station aquicole de Pointe-au-Père. Enfin, je souhaiterais

remercier ma famille et mes amis pour leur soutien tout au long de mes études. Merci de

m'avoir permis de réaliser mes projets bien que loin de la maison ... Ce fut l'occasion de

venir découvrir le Québec!

Cette maîtrise a pu être réalisée grâce à l'aide financière du FQRNT et de la SODIM. De

plus, j'ai bénéficié du support financier de l'Institut des sciences de la mer de Rimouski .

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AVANT-PROPOS

Le chapitre II de ce mémoire de maîtrise intitulé « Resistance of charr to opportunistic

infections in seawater» est présenté sous la forme d'un article. Lors de la soumission,

l'article sera séparé en deux papiers distincts. L'un portera sur l'identification de l'agent

pathogène responsable d'infections opportunistes en milieu marin chez les ombles, alors

que l'autre sera basé sur la relation entre la génétique et les différences de résistance

interfamiliales des ombles face aux infections opportunistes en milieu marin.

111

RÉSUMÉ

Cette étude vise à éclaircir la relation qui existe entre la génétique et la résistance naturelle des ombles face aux infections opportunistes présentes en milieu marin, améliorant ainsi leur survie dans cet environnement. Afin de rencontrer cet objectif, 25 familles demi-frère ont été produites à l'automne 2003 : 15 familles d'ombles de fontaine (Salvelinus fontinalis) , 5 familles d'ombles chevaliers (Salvelinus alpinus) et 5 familles hybrides d'ombles chevaliers - ombles de fontaine . L'identification du génotype des parents utilisés pour les différents croisements a permis de mettre en évidence la présence de 21 allèles du complexe majeur d'histocompatibilité (CMH) de classe npl. La majorité des parents (37sur 43) étant hétérozygotes (porteur de deux allèles différents), une gamme importante de combinaisons alléliques devrait être retrouvée dans la progéniture. Durant l' été 2005, les ombles âgés de 1+, ont été transférés en eau de mer, puis soumis à des stress aigus afin d 'évaluer la résistance aux infections opportunistes. Pour la croissance, des interactions significatives ont été trouvées entre les types de croisements et les divers temps d' échantillonnages. Étant en présence de deux types d'hybrides intra et interspécifiques, des comparaisons intraspécifiques impliquant les familles AA, AR, RR et des comparaisons interspécifiques impliquant les familles AA, CF, CC ont été effectuées. Au-delà de l' importante hétérogénéité de croissance en eau de mer (intra- et interspécifique), une tendance générale se dégage. Les familles ayant les meilleures croissances en eau douce sont celles qui présentent la meilleure croissance en eau salée. De plus, des différences interfamiliales (intra- et interspécifiques) de résistance/susceptibilité ont été obtenues. Les familles anadromes et résidentes S. fontinalis sont les plus susceptibles aux infections, alors que les hybrides anadromes interspécifiques S. alpinus - S. fontinalis sont les plus résistants. Sur les 25 familles transférées en eau de mer, cinq d' entre elles sont hautement résistantes, aucune mortalité n' ayant été enregistrée. Des frottis des lésions présentes chez les poissons affectés ont permis d' amorcer des analyses microbiologiques. Les résultats des analyses microbiologiques ont été confirmés par des analyses moléculaires et ont ainsi permis l' identification de l'agent pathogène en cause : Flexibacter maritimus.

IV

TABLE DES MATIÈRES

REMERCIEMENTS .. ... .... .... ......... ............ ............. .. .... ...... .... .... .... ...... .... .... ... .................. . i

AV ANT -PROPOS ..... .... .. .. ... ........ .... .... .... .. ... .... .... .... ...... ... ........... ................ ...... ............. . ii

RÉsUMÉ .. .. ...... ............ .. .... .. ...... ...... .. ... ... .... .......... ... ..... .... .... .... .... ... ............. ............. ..... iii

TABLE DES MATIÈRES .. .... .... ....... .... .. ....... ...................... ... .... ... .... ... .... ... ... .. ... .... .... .. ... iv

LISTE DES TABLEAUX .......... ........ .... .... ... .................. ... ..... ....... .... ........ ....... ..... ... ... ..... vi

LISTE DES FIGURES .......... .. ....... .... .. ...... ... .. .. .. ... .. ..... ... .. .. ............ .... ..... ........ ... .. ... ....... vii

LISTE DES ANNEXES .................... .... .. .. ..... ........ ........ ... .. .. ... .... ... ... ....... .... ...... ..... ......... ix

1. CHAPITRE 1. INTRODUCTION GÉNÉRALE ..... ..... .... ..... .. .. .... .. .. ... .... ... .... .... .... ... ...... 1

1.1. Le système immunitaire .... ... .... ..... ....... ... ..... .. .. .... ..... ..... .... .. ... .. .. ..... ... ..... ... .... ..... 1

l.2. Le complexe majeur d'histocompatibilité ..... ......... ... .. ..................... ...... ... ... .. ... ... . 2

1. 3. Les maladies bactériennes en aquaculture ...... ..... .... ...... ... .. .... ... ... ..... ...... ......... .... . 4

1.4. Le stress et la résistance aux maladies ... ............. .... ..... .. .. .......... ...... .. ........ .. .. ........ 5

1.5. Problématique et objectifs .... .... ......... .... .. ...... ........ ...... ...... ... ... .... .... ........ ............. 6

II. CHAPITRE II. RESISTANCE OF CHARR (Salvelinus fontinalis) AND (Salvelinus

alpinus) TO OPPORTUNISTIC INFECTIONS IN SEAWATER ....... ................... .... .... ..... 9

2.1. Introduction ........... ............... ..... .. ... .. .. ............................... ............ .... .... ... .. .......... 10

2.2. Material and methods ... .. ....... ... .. .. ......... .... .... ... .... ..... ... ......... ... ... ..... ..... ... .... ... ..... 13

2.2.1. Fish ............ ... ..................... .. .. .. .............. ........... ... ... .... ..... .. ... .. .. ..... .. ... .. .. .. 13

2.2.2. Parental genotypes ofMHC class II (region Pl) alleles ......... .. ........ .. ..... ... 16

2.2.3. Identification of pathogens ...... ................ ...... ...... .... .. ...... .... ... ........ .... ... .... 20

v

2.2.4. Stress exposure ....... .... .. .. .. ....... ... .. .......... ... .... ..... .... .... .... .... ...................... 22

2.2.5. Statistical analysis .... .... .... ... .. .. .. .. ..... ... .. .. ................ ... ............. .................. 23

2.3 . Results .. ...... ............. .. ... ....... ...... .... ............. .... ... .... .... ......... .. ..... .... .... ...... ........... . 24

2.3.l. Parental genotypes ofMHC class II (region Pl) alleles .......... .. .......... .... ... 24

2.3.2. Identification of pathogens ..... .. ... .. ... ... .... ... ....................... .. .... ....... .. .. .. ... .. 29

2.3 .3. Stress exposure .. .. ......... ..... ........ .......... .. .. .. .. .. .. .. ...... .... ..... .. ... ..... ... ........... 32

2.4. Discussion ......... ........................... .. ..... ... ....... ............... .... ....... .. .............. ....... .. .... 47

III. DISCUSSION GÉNÉRALE ......... ............ ......... .. ...... .. .. .. .. ........ .. .. .... .... .. .. ... ... .. .. ....... 52

IV. CONCLUSION GÉNÉRALE .. .. ................. ..... ..................... .... .. .. .... .. ..... .... ........... ... 56

LISTE DES RÉFÉRENCES .............. ... ..... ........ .............................. ......... ... ....... .............. 57

Vi

LISTE DES TABLEAUX

Tableau 1. Salvelinus fontinalis and Salvelinus alpinus - Salvelinus fontinalis cross-

breeding ... ...... .... .... .......... ... ... .... .... ... .. ..... .. ... .. ... .... ..... .... ... ..... ... .... ... .... .. .. ..... ... ....... 15

Tableau 2. Allele frequencies from broodstock ...... .. ...... .. .... .... ...... .. .... .. .... ... .. .. .. .. ........... 30

Tableau 3. Parental and expected progeny genotypes for Salvelinus fontinalis and

Salvelinus alpinus .... ... ........ ........... .......... .. .. ..... .... .... .... ... .... ....... .... .... ...... .......... ... ... 31

Tableau 4. Two-way ANOV A results ... .. ............ .. .. ................. ... .. ... ..... .. ..... .. .... .. .... .. ....... 35

Tableau 5. Condition factor of intraspecific Salvelinus fontinalis (AA, AR, RR) and

interspecific Salvelinus alpinus - Salvelinus fontinalis (AA, CF, CC) families during

summer time .... ......... ... ..... .... .... .......... ... ....... .......... ............ .... .. ..... .. .. ... ..... .. .. ......... .. 40

VIl

LISTE DES FIGURES

Figure 1. Exon 2 sequences of the MHC class IIJ3l alleles in Salvelinus alpinus and

Salvelinus fontinalis ................. .... .... ............. ........ ... ..... ......... ... ... .... ... .... .. .. .... ... .... .. 25

Figure 2. Amino-acid sequences for MHC class IIJ3l alleles in Salvelinus alpinus and

Salvelinusfontinalis ............................... .... ........... .... .... .... .... .... ... ................... ......... 27

Figure 3 : Silver-stained SSCP gel of the MHC class II pl from Salvelinusfontinalis and

Salvelinus alpinus ... ...... .. ..... ... .. .... ... .. .......... ... ..... .. ............ ... ............ ..... .. .......... .......... .29

Figure 4 . Nested PCR products obtained from sick fish .................................................... 34

Figure 5. Fish weigth of the intraspecific Salvelinus fontinalis families (AA, AR, RR)

during summer time ...................... ..... .. .. .............................................. .. ................... 38

Figure 6. Fish length of the intraspecific Salvelinus fontinalis families (AA, AR, RR)

during summer time .......... .... ... ......................... .... ......... .. .. .... ..... ..... ....... ..... ........... 39,

Figure 7. Fish weigth of the interspecific Salve lin us alpinus* Salvelinus fontinalis families

(AA, CF, CC) during summer time ........ .. ......................................... .... .................. 41

Vlll

Figure 8. Fish length of the interspecific Salve lin us alpinus* Salvelinus fontinalis families

(AA, CF, CC) in September.. ..... ..... ......... .. ......... .... ... ... ... .. .. .. ..... ...... ..... .... .. .... .... ... 42'

Figure 9. Cumulative mortalities in anadromous Salvelinusfontinalis families (AA) during

summer time ......... .... .... .. .. ... ..... .... .... ... .. .... .. .. .... .... .... ... ..... ........... ..... .. .... .. .. .... .... .... . 44

Figure 10. Cumulative mortalities in resident Salvelinus fontinalis families (RR) during

summer time .. .... ....... .. .. ................ .... ..... .... ... ....... ...... .. .. .. ......... ....... ..... ... .. ..... ...... .... 44

Figure Il . Cumulative mortalities in anadomous-resident Salvelinus fontinalis families

(AR) during summer time ..... ....... .. .. .... .... .... ... .... ............ ...... .. ... .... .. .. ...... .. ....... .. .. ... . 45

Figure 12. Cumulative mortalities in anadromous Salvelinus alpinus * Salvelinusfontinalis

families (CF) during summer time ... ........ .... .. .. .......... .............. ... ...... ... .. .... ... ....... .. ... 45

Figure 13 . Cumulative mortalities in anadromous Salvelinus alpinus families (CC) during

summer time ............... ... ... .... .... .... ... .. .... ....... .. ... .. .... ..... .. ........ .... ...... ... ... ... .... ... ........ 46

IX

LISTE DES ANNEXES

Annexe 1. Exemple du "liability/threshold model" pour la famille résidente Salvelinus

fontinalis R8 ....... .. .. .. .. ... ...... .... .. ... .. .... ....... .... .. .. .. ... ......... .... ........ ... ..... .... .... ... .. ........ 66

Annexe 2. Tableau présentant, pour les familles intraspécifiques Salvelinus fontinalis et

interspécifiques Salvelinus alpinus - Salvelinus fontinalis, les proportions de

populations pour chacune des quatre classes phénotypiques .............. .. ...................... 67

1. CHAPITRE 1

INTRODUCTION GÉNÉRALE

1.1 Le système immunitaire

1

Le système immunitaire assure la protection de l'organisme par discrimination entre

le soi et le non-soi (Bensa, 2003). La pénétration de microorganismes (antigènes) induit une

réponse immunitaire adaptative, qui met en jeu deux types de réponses: des réactions non

spécifiques et des réactions spécifiques.

D'action rapide, l'immunité innée constitue la première ligne de défense contre les

infections, capable de limiter la prolifération des agents pathogènes dans l'organisme. Cette

réponse non spécifique s'exerce de la même façon quel que soit l' antigène. Une réaction

inflammatoire située au niveau du foyer infectieux, attire sur place les cellules

phagocytaires (Kollner et al., 2002). Les macrophages, cellules présentatrices d'antigènes,

phagocytent l' agent pathogène et dégradent ses macromolécules antigéniques en peptides

par digestion dans le phagolysosome. C'est cette forme modifiée de l'antigène qui, associée

aux molécules du complexe majeur d'histocompatibilité (CMH), génère les ligands

spécifiques des récepteurs antigéniques des lymphocytes T, capables de déclencher une

réaction immunitaire spécifique (Janeway, 2001). En effet, si l' immunité innée n'est pas

suffisante, les macrophages interagissent avec les lymphocytes afin d'activer l' immunité

acquise, qui est spécifique et donc plus efficace. Elle se manifeste sous deux formes :

l' immunité à médiation humorale qui fait intervenir les lymphocytes B, responsable de la

2

production d'anticorps et l'immunité à médiation cellulaire qui dépend de la reconnaissance

spécifique de l'antigène par les lymphocytes T (Nakanishi et al. , 1999).

1.2 Le complexe majeur d'histocompatibilité

Les molécules du complexe majeur d' histocompatibilité (CMH) sont impliquées

dans de nombreux aspects de la reconnaissance immunitaire (Bernatchez et Landry, 2003).

Contrairement aux cellules B qui lient directement l'antigène, les cellules T ne

reconnaissent l'antigène qu'en association avec les molécules du CMH de classes 1 et II. Ces

deux classes de glycoprotéines membranaires de structure très similaire, mais de fonction

distincte, ont toutes deux un rôle crucial à jouer dans l' apprêtement des peptides

antigéniques aux cellules T (Grimholt et al. , 2000).

Les molécules de classe I, exprimées à la surface de pratiquement toutes les cellules

nucléées, présentent des antigènes peptidiques endogènes (intracellulaires) aux cellules Tc

(cytotoxiques). S'il s' agit d 'un peptide étranger, un lymphocyte T cytotoxique (CD8+) se

fixera au complexe CMH-peptide et finira par tuer la cellule infectée. Dans le cas des

molécules de classe II, exprimées essentiellement sur les cellules présentatrices d' antigènes

(macrophages, cellules dendritiques et cellules B), les peptides antigéniques exogènes

(extracellulaires) sont présentés aux cellules TH ( auxiliaires) (Trevor, 1996). Les

lymphocytes T auxiliaires (CD4+) ne tuent pas directement leurs cellules cibles, ils se

divisent dans un premier temps, augmentant ainsi leur nombre, puis sécrètent des cytokines.

3

Celles-ci ont un rôle de régulation des phénomènes immunitaires; soit elles inhibent

l' antigène, soit recrutent et stimulent d'autres cellules qui se joignent à la réponse

immunitaire. Les molécules de classe II sont nécessaires pour que les cellules T

communiquent avec les macrophages et les cellules B (Bernatchez et Landry, 2003).

Les molécules du CMH de classe II sont composées de domaines externes, d'un

segment transmembranaire et d'un segment d'ancrage cytoplasmique. Ces glycoprotéines

membranaires forment de façon non covalente un hétérodimère de chaînes a et J3,

respectivement de masse moléculaire 34 et 28 kDA . Chaque chaîne est composée de deux

domaines extracellulaires (al et a2; J31 et J32) . Les domaines a2 et J32 proches de la

membrane (proximaux), présentent une homologie de séquence avec la structure du

repliement immunoglobulinique; c'est pour cette raison que les molécules de classe II du

CMH sont classées dans la superfamille des immunoglobulines (Sü1tmann et al. , 1994). Le

polymorphisme de ces molécules est principalement situé dans les domaines a 1 et J31 ,

éloignés de la membrane (distaux), qui forment la cavité de liaison au peptide. Cette

variation structurale représente la base fondamentale permettant la spécificité antigénique

(Bouillon et Mourad, 2003).

Les gènes du CMH font partie des gènes les plus polymorphiques connus jusqu ' ici,

avec de multiple loci et un nombre considérable d'allèles pour chaque locus donné

(Grimholt et al. , 2003). Cette diversité augmente le nombre de peptides antigéniques

pouvant être reconnus, ce qui s' avère avantageux pour l' espèce. Différentes études ont

4

montré que les gènes de classes 1 et II sont situés sur des groupes de liaison génétique

différents et que le saumon Atlantique possède un seul locus codant pour le CMH de classe

1 et un seul codant pour le CMH de classe II (Grimholt et al., 2002; Stet et al., 2002). Les

gènes de classe II sont organisés en une série d'exons et d'introns qui reproduisent la

structure en domaines des chaînes a et p. Notre étude porte sur le domaine Pl ,

correspondant à la région la plus polymorphique, codée par l'exon 2 (Ono et al., 1993).

1.3 Les maladies bactériennes en aquaculture

De nombreuses maladies bactériennes sont liées à des facteurs qUi stressent ou

affaiblissent les poissons, les rendant ainsi moins résistants aux infections. Elles peuvent se

classer en deux catégories, celles qui affectent des zones spécifiques du corps (infections

localisées) et celles qui affectent le corps en entier (infections systémiques). De nombreuses

pertes causées par les infections bactériennes systémiques ont été répertoriées dans les

élevages de salmonidés à travers le monde (Avendafio-Herrera et al., 2005). La furonculose

(agent pathogène Aeromonas salmonicida), la maladie de la bouche rouge (agent pathogène

Yersinia ruckeri), et la maladie bactérienne du rein (agent pathogène Renibacterium

salmoninarum) sont identifiées à la fois en eau douce et marine. La vibriose (agent

pathogène Vibrio anguillarum), et la vibriose en eau froide (Vibrio salomonicida) touchent

uniquement les élevages en eau de mer contrairement à l'aéromonose (agent pathogène

Aeromonas hydrofila) qui n'apparaît qu' en eau douce (Trevor, 1996). De nombreux agents

pathogènes (Cytophaga sp, Flavobacterium sp et Flexibacter sp) appartenant anciennement

5

au genre Flavobacterium-Cytophaga sont regroupés dans le phylum Cytophaga-

Flavobacterium-Bacteroides (Bernadet, 1997) et causent également des infections

systémiques à la fois chez des poissons cultivés en eau douce ou chez ceux produits en eau

marine. L'espèce Flexibacter maritimus a été récemment reclassée dans le genre

Tenacibaculum, appartenant à la famille des Flavobacteriaceae (Suzuki et al. , 2001) .

1.4 Le stress et la résistance aux maladies

Les poissons d'élevage sont soumis à des situations de stress répétés qui peuvent

aVOIr des conséquences négatives plus ou moins importantes sur la croissance, la

reproduction ou la résistance aux agents pathogènes (revue de Pickering, 1992). Le stress et

la résistance aux maladies sont liés, mais les mécanismes de cette relation sont

extrêmement complexes (Ellis, 1981) et jusqu'à récemment peu connus. Des paramètres de

tolérance aux stress ont été trouvés et associés avec la résistance des poissons aux maladies

(Fevolden et al., 1991 ; 1993 ; 1994; 1999). On parlera de résistance pour la réaction à une

modification brusque et intense, et de tolérance pour l'adaptation à des modifications

durables des caractéristiques du milieu (Vandeputte et Prunet, 2002). Le cortisol est

considéré comme étant un bon indicateur de stress chez le poisson (Wendelaar Bonga,

1997). Sa libération est à court terme favorable pour le poisson (mobilisation de l'énergie,

rétablissement de l'homéostasie ... ) mais a par contre des effets négatifs sur différentes

fonctions physiologiques (reproduction, croissance, immunité ... ) lorsqu'elle se maintient

dans le temps (stress chroniques ou répétés) (pottinger et Pickering, 1997).

6

1.5 Problématique et objectifs

L'émergence de nouvelles filières concernant le développement d'une production

d'ombles chevalier (Salvelinus alpinus) et d'ombles de fontaine (Salvelinus fontinalis) en

eau salée, se heurte à un problème de santé. De nombreux travaux effectués sur les ombles

anadromes à la station aquicole de Pointe-au-Père ont montré que différents stress

pouvaient provoquer des lésions de types opportunistes (localisées sur les branchies, les

flancs et la queue) chez les animaux élevés en eau de mer (Audet, communication

personnelle). Les analyses bactériologiques effectuées par le MAP AQ indiquaient des

infections causées par des myxobactéries. Toutefois, une analyse plus poussée s' avère

nécessaire puisqu'à l' été 2004, les résultats d' identification des pathogènes étaient

différents de ceux obtenus les années précédentes pour des symptômes similaires. De plus,

ces lésions n'affectent pas les différentes familles de la même façon, certaines sont peu ou

pas touchées alors que d 'autres peuvent être décimées (Bastien, communication

personnelle). Il semblerait donc qu'il existe une relation entre les caractéristiques

génétiques et la résistance.

Une étude portant sur le saumon Atlantique a permis d' établir pour la première fois

une corrélation entre la variation allélique du complexe majeur d' histocompatibilité de

classe II et la résistance à un agent pathogène bactérien Aeromonas salmonicida (Langefors

et al. , 2001). L'association entre la résistance aux maladies infectieuses, de nature virale et

bactérienne, et le polymorphisme du CMH classes 1 et II chez le saumon Atlantique a été

confirmée (Grimholt et al. , 2003). D'Autray Tarte (2004) a étudié si certains allèles du

7

CMH classe II, domaine ~l chez l'omble de fontaine (Salvelinus fontinalis) (souche

Rupert) étaient liés à une résistance accrue face à une infection de furonculose causée par

l' agent pathogène Aeromonas salmonicidae. Cinq allèles (A, B, D, E et F) provenant de 62

géniteurs ont été répertoriés et ont permis d'effectuer des croisements générant 24 familles

de génotype différents. Cependant, aucune famille n' avait pu être constituée à partir des

allèles E et F car les géniteurs porteurs de ces allèles n'étaient pas prêts pendant la période

de frai (d'Autray Tarte, 2004). Il semblerait qu'une corrélation soit observable entre le

nombre de copies de l' allèle A et le temps accru de survie post infection. Cette corrélation

se retrouverait également avec l'allèle B mais la résistance serait moindre que celle

procurée par l' allèle A (d'Autray Tarte, 2004). Au cours de son projet, d'Autray Tarte avait

également répertorié quatre allèles (P, H, 1 et L) du CMH classe II, domaine ~l provenant

de 68 géniteurs d'ombles de fontaine (Salvelinus fontinalis) de la souche Laval. L'étude de

Nassif (2006) visait le même objectif mais pour les allèles du CMH classe l, domaines al et

a2 . À partir des 24 familles d'ombles de fontaine (souche Rupert) générées par d'Autray

Tarte, deux allèles (A et B) ont été identifiés concernant le domaine al et cinq allèles (A, B,

C, D et E) concernant le domaine a2. En se basant sur ces études, l'hypothèse selon

laquelle certains allèles du CMH pourraient prédisposer certains individus à une résistance

accrue aux infections améliorant leur survie en milieu marin sera vérifiée pour les ombles

(Salvelinus fontinalis) et (Salvelinus alpinus).

Des croisements inter et intraspécifiques de familles en demi-frère, permettant

d'observer l' effet maternel, ont été générés à partir d'ombles chevalier (Salvelinus alpinus)

anadromes (souche Fraser) et d'ombles de fontaine (Salvelinus fontinalis) anadromes et

8

résidentes (souche Laval). Des biopsies non-létales ont été effectuées sur les géniteurs afin

de constituer une banque de données génétiques. Les différentes familles ainsi produites ont

été soumises à des essais de résistance en milieu marin dans le but de mesurer les

différences inter-croisements et ainsi d' identifier les combinaisons alléliques du CMH

menant à une meilleure réponse immunitaire. Au cours de cette étude, trois objectifs ont été

poursuivis: 1°) l'identification moléculaire des agents pathogènes opportunistes qui

affectent les élevages d'ombles en milieu marin; 2°) l'identification du génotype des parents

des différents croisements effectués pour les allèles pl du complexe CMH; 3°) la mesure

des différences inter-croisements de résistance aux infections de type opportuniste par des

mesures d'occurrence et de suivis de mortalités. L' hypothèse nulle, « HO : il n'y a pas de

différences inter-croisements pour la résistance aux maladies », a alors été vérifiée.

Afin de vérifier si une résistance accrue peut être associée à la présence ou à

l'absence de certains allèles, et d' examiner si les différences de résistance sont liées à des

différences alléliques au niveau de la région p l , ce projet devra être suivi d'une étude de

corrélation entre les résultats obtenus pour les objectifs 2 et 3, permettant de cibler les

progénitures résistantes versus non-résistantes et du génotypage des progénitures.

ll. CHAPITRE II

RESISTANCE OF CHARR (Salvelinusfontinalis) AND (Salvelinus alpinus)

TO OPPORTUNISTIC INFECTIONS IN SEA W ATER

9

10

2.1 Introduction

Fish protect themselves against pathogenic microorganisms by an immune system

comparable to that of humans and other vertebrates (Anderson, 1990). The first line of

defence is based on non specific immune mechanisms. If a pathogen penetrates into the

organism, the resulting inflammation attracts phagocytic cells that destroy the invader. This

natural resistance is normally effective enough to protect fish from infectious diseases until

specific immune responses are being induced (Kollner et al. , 2002). If the non specific

response is unsuccessful, the major histocompatibility complex (MllC) cannot activate the

specific immune response by the presentation of antigenic peptides to T cells. To be

recognized, a foreign antigen must be degraded into smalt antigenic peptides that form

complexes with class 1 or class II MllC molecules. These molecules are structurally and

functionally distinct cell surface glycoproteins involved in antigen presentation to T -cells.

Class 1 molecules, which are expressed on the surface of all nucleated cells are involved in

the presentation of endogenously derived peptides to CD8-positive (cytotoxic) T

lymphocytes. On the other hand, class II molecules, expressed mainly in the antigen-

presenting cells (B lymphocytes, macrophages, and activated T cells) present exogenously

derived peptides to CD4-positive (helper) T lymphocytes (Iwama and Nakanishi, 1996).

The MllC genes represent sorne of the most polymorphic genes, with multiple loci

and a considerable number of alleles at each given locus (Grimholt et al. , 2002). They play

an important role in disease resistance to pathogens by their efficacy to present a wide

11

variety ofantigenic peptides derived from pathogens (Van Muiswinkel et al., 1999). It was

recently found that the teleostean class 1 and class II genes reside on different linkage

groups. Furthermore, several studies in salmonids have shown that each class contains one

major and dominantly expressed locus (Grimholt et al., 2003) .

Class II MHC proteins are heterodimers composed of two non-covalently linked

glycoproteins, the a (34 kDA) and ~ (28 kDA) chains, which are encoded by separate class

II genes organized into a series of exons and introns. Each chain consists of two external

domains (a 1 and a2 or ~ 1 and ~2), a connecting peptide, a transmembrane segment, and a

cytoplasmic tail. The a2 and ~2 domains are charracterized by an immunoglobulin fold,

which places the MHC molecules in the family of immunoglobulin-like molecules. The

membrane-distal do main composed by a 1 and ~ 1 domains is involved in the interaction

with peptides and constitutes the peptide binding region (PBR) (Sültmann et al., 1994). The

ability ofboth class 1 and II genes to face various pathogens is believed to be mainly related

to sequence variation among MHC alleles in the peptide binding region PBR (Bernatchez

and Landry, 2003). Variation in exon 2, which codes for ~ 1 domain, is responsible for most

of the polymorphism observed in the class II genes; most of it is concentrated in the codons

specifying the PBR (Sültmann et al., 1994).

ln aquaculture, stress factors increase fish susceptibility to opportunistic infections

by compromising the functioning of the immune system (Kollner et al., 2002). Different

studies realised on anadromous brook charr in our laboratory showed that stress factors

12

may affect fish raised in salt water by opportunistic lesions (Audet, personnal

communication). However, observations from a genetic study indicated that these lesions

are not present in aIl families; sorne are atTected whereas others are not (Bastien, personal

communication). A study realised on Atlantic salmon by Langefors et al. (2001) showed a

correlation between MHC class II allelic variation and resistance to the bacterial pathogen

Aeromonas salmonicida. This association between resistance towards infectious diseases

and MHC class 1 and II polymorphism was confirmed by Grimholt et al. (2003). In

Salvelinus fontinalis , Rupert strain, d' Autray Tarte (2004) and Nassif (2006) respectively

studied MHC class II (domain Pl) and MHC class 1 (domains al and (2) . Five alleles (A,

B, D, E, F) were found for the MHC class II Pl , two alleles (A, B) for the MHC class 1 al ,

and five alleles (A, B, C, D, E) for the MHC class 1 a2. In addition, d 'Autray Tarte also

identified four alleles (P, H, l , L) for the MHC class II (domain Pl) in the Laval strain.

Based on the se different studies, the hypothesis that sorne MHC alleles should

predispose sorne charr to a better resistance to a bacterial pathogen was verified. Different

cross-breeding between Arctic charr (Salvelinus alpinus) and brook charr (Salvelinus

f ontinalis) were planned and samples were collected to identify alleles present in

broodstock. Stress resistance of the ditTerent families was challenged in order to measure

differences in sensitivity between cross-breeding and identify allelic combination leading to

a better immune response. The aims of the present study were: 1) to identify opportunist

pathogens that affect charr in salt water; 2) to identify the parental genotype of MHC class

13

II~ alleles for resistance to an infectious disease by focusing on exon 2; and 3) to compare

familial resistance to this pathogen.

2.2 Material and methods

2.2.1 Fish

Intra and interspecifics cross-breeding between anadromous Arctic ch arr, Salvelinus

alpinus, (strain issued from the Fraser River) and both, anadromous and resident brook

charr, Salvelinus fontinalis, (strains issued from the Laval River), were realised in faH 2003

at the Station aquicole de Pointe-au-Père, Québec (48°31 'N; 68°29 'W). Five half-sib

families for each following crosses were produced: 1) S. fontinalis

anadromous*anadromous, anadromous*resident, resident*resident; 2) S. alpinus

anadromous*anadromous; 3) anadromous hybrid S. alpinus*S. fontinalis (female is

indicated first) (Table 1). The twenty-five families had their own marking (left, right or

both pelvic fins cut) . In May 2005, families were reduced in number to 150 individuals per

family (at the exception of one family CF7: only 90 fish) . Fish were sampled randomly.

They were raised into ten different 500 L tanks (fish from three families per tank) supplied

with a constant flow (10 L min-l) of recirculating water. Fish were fed with commercial

pellets (HiPro Fretin Corey Fry Fish Feed, Canada) at a rate of 2.5% (from July 2004 to

September 2004) or 1% (from September 2004 to September 2005) body weight per day,

and raised under natural photoperiod, temperature, and salinity conditions. Weight and

14

forklength measurements were realised on 50 individuals per family according to the

following schedule: July, and September 2004; February, April, July, and September 2005 .

Before measurements, fish were anaesthetized with 3-aminobenzoic acid ethyl ester (1 .2

!lmol L-1 ofMS222) (Sigma-Aldrich, Germany), and their markings checked.

15

Cross Family Dam Sire AA anadromous S. fontinalis Al J062 BC04l *

X A2 J075 BC045* anadromous S. fontinalis A3 J15l BC043

A4 J152 BC044 A6 B014 BC046*

AR anadromous S. fontinalis ARI J157 V002* X AR2 J153 VOOO*

resident S. fontinalis AR3 J056 VOOI AR4 J159 V060* AR5 J155 V005*

RR resident S. fontinalis R2 R005 V005* X R3 ROOO V002*

resident S. fontinalis R6 R053 V062 R7 R049 V060* R8 R058 VOOO*

CF anadromous S. alpinus CFI BC054 BC046* X CF2 BC053 BC04l *

anadromous S. fontinalis CF5 BC040 BC057 CF6 BC039 BC047 CF7 BC070 BC045*

CC anadromous S. alpinus C2 BC06l BC060 X C4 BC066 BC065

anadromous S. alpinus C5 BC055 BC062 C6 BC056 BC068 C9 BC069 BC079

Table 1. Salvelinus fontinalis and Salvelinus alpinus - Salvelinus fontinalis cross-breeding. Female is indicated first. Asterisks indicate that the same sire was used for two crosses in half-sib families.

16

2.2.2 Parental genotypes ofMHC class II (region /31) aile les

DNA extraction

Adipose fins from the parental Arctic and brook charr were collected for genetic analysis.

DNA was extracted from these 43 ethanol-preserved adipose fins using the Montage BAC96

Miniprep Kit (Millipore, USA). Each sample was disposed on 96-well culture block and

received 500llL of lysis butTer (1 % sodium dodecyl sulfate (SDS), 50mM Tris-HCl, pH

8.0, O.lM EDTA, pH 8.0, and O.lmg mL- l protease K) before being incubated ovemight at

37°C with constant agitation. DNA was isolated from protein residues by following the

Millipore proto col. Then, purified DNA resuspended in 351lL of 10mM Tris-HCl, pH 8.0,

was stocked at -20°C.

Polymerase chain reaction amplifications

To amplify the MHC class II W1 region) sequences from brook and Arctic charr, a forward

and reverse primers located on the beginning and the end of exon 2 were used. A sense

primer TVS 4501 (5'-CCTGTATITATGITCTCCTITC-3') was selected from the study of

Langefors el al. (2001), whereas the corresponding antisense primer SP4502 (5 '

TAAGTGITGCTACGGAGCC-3 ' ) designed by d'Autray Tarte (2004) was used. Nevertheless,

for seven samples, the antisense primer ALI002 (5'-CACCTGTCTIGTCCAGTATG-3 ') used by

Langefors et al. (2001) was preferred due to better results obtained with SP4502. The

amplification was carried out in the DNA Thermal Cycler Biometra T Personnal under the

following conditions: denaturation for 3 min at 94°C, 34 cycles of denaturation for 30 sec at

17

94°C, annealing for 30 sec at 47°C, extension for 1 min at 72°C, followed by a final

extension for 10 min at 72°C. The 50llL polymerase chain reaction (PCR) mixture

contained: 3111 ofDNA, 1111 of each primer (5pmol), 0.5111 ofTaq polymerase Expand High

Fidelity PCR system (Roche, Germany), 5111 of Expand Taq reaction buffer (10X), 1111 of

Expand Taq MgCh (25 mM), 1111 ofdNTP mix (lOmM). When run on a 1% agarose gel, the

PCR products showed one distinct band corresponding to 315 bp with SP4502 or 296 pb

with AL1002.

Analysis of Single-Stranded Conformation Polymorphism (SSCP)

In order to detect whether or not DNA fragments from the 43 samples were identical in

sequence, we used the SSCP technique. It allowed us to detect DNA polymorphisms, which

result in change in mobility of the single strands on gel electrophoresis. At the end of the

PCR cycling, 10111 of each PCR products was mixed with 10111 of formamide loading

(0.25% xylene cyanol and 0.25% bromophenol blue). Samples were denatured for 3-4 min

at 95°C in a water bath and were placed directly on ice. From this preparation, 15111 was

loaded onto 10% non-denaturing polyacrylamide gels. Gels were composed of 2.5ml of

acrylamide (49 : 1) (40%), 1.25ml of glycerol (40%), 1 ml ofTBE (10X), 5.25ml of distilled

water, 100111 of ammonium persulfate (10%), and 20111 Temed. The TBE (IX) running

buffer was prepared the night before and was refrigerated at 4°C. The electrophoresis tank

as weIl as the TBE were maintained at 4°C during the migration. The conditions for

electrophoresis (lOOV for 10 min, then 200V for 2 hours at 4°C) were standardized for

18

optimal resolution of bands. At the end of the migration, gels were silver stained according

to the protocol ofBudowle et al. (1991) .

Cloning

SSCP analyses allowed to group individuals with similar band patterns. DNA of individuals

from each group was cloned and sequenced. After purification by chloroform extraction,

PCR products were ligated to the pT7Blue-2 Vector and cloned into Escherichia coli

DHIOB competent cells according to the Perfectly Blunt Cloning Kits Manual (Novagen,

New England). We used the chromogenic substrate 5-bromo-4-chloro-3-indolyl-~-D

galactoside (X-gal) (20mglml in dimethylformamide) to visualize recombinant colonies by

blue/white screening; bacteria carrying recombinant plasmids form white colonies. To

confirm the presence of the appropriate insert, ten white colonies, picked with a sterile tip

conserved in 2YT preparation at 4°C, were firstly amplified by PCR and depending on the

PCR product size, were brought under cultivation overnight. Then, plasmid DNA was

extracted by mini preparation SDSlNaOH following the proto col described by Birnboim and

Doly (1979).

Digestion

The orientation of the insert was determined by the analysis of the digestion profile,

realised with the restriction enzyme Alu 1 (New England Biolab). Digestion was realised

with IOfll of DNA extracted by minipreparation, 2fll of buffer #2 (IX) (New England

19

Biolab), and 0.5J.l.I of Alu I. Samples were incubated one hour at 37°C, before being loaded

onto 20% polyacrylamide gels. Gels were composed of6.67ml ofacrylamide (29 :1) (30%),

Iml of TBE (10X), 2.33ml of distilled water, 100J.l.I of ammonium persulfate (10%) and

20J.l.I of Temed. The migration conditions were the same as described with SSCP. At the

end of the migration, gels were immerged 30 min in 100 ml of distilled water with 5J.l.I of

ethidium bromide. Bands were visualised with Chemi Imager 5500 (Alpha Innotech

Corporation). Two inserts with different orientations were chosen for each sample and were

purified with Sephaglas™ BandPrep Kit following the manufacturer' s instructions

(Amersham Pharmacia Biotech, UK).

Sequencing and identification of alleles

Our selected samples were sent to the sequencing services provided by the immunogenetic

department at the Notre-Dame campus of the Centre hospitalier de l'Université de Montréal

(CHUM) , which used an ABI 3100 DNA sequencer with 16 capillaries. Ali sequence

alignments were carried out using ClustalW (European Bioinformatics Institute) and amino

acids sequences were obtained using MEGA version 3.1 (Kumar et al., 2004). From the

difTerent sequences found, our difTerent alleles were determined. Another PCR

amplification from parental DNA was realized. Purified PCR products were sent to the

sequencing services in order to determine genotypes and to confirm the difTerent alleles. By

analysing the different chromatograms using Sequencher (Gene Codes Corporation),

heterozygotes versus homozygotes individuals were determined.

20

2.2.3 Identification ofpathogens

Media preparation

Different selective culture medium were prepared and stocked at 4°C in order to facilitate

the isolation and identification of the pathogens responsible of opportunists lesions

affecting charr in salt water. Marine agar (MA) was used to detect total bacteria from water

sampi es, TCBS for the detection of Vibrio spp, Anacker and Ordal agar (AOA) prepared

with seawater (contained in grl: 5g tryptone, 0.5g yeast extract, O.2g beef extract, O.2g

sodium acetate, 15g Agar, IL of salt water, pH: 7.2-7.4) for the detection of

Flavobacterium spp (Anacker and Ordal, 1959) and Flexibacter maritimus Medium (FMM)

also prepared with seawater (contained in grl : 5g peptone, 0.5g yeast extract, O.Olg sodium

acetate, 15g agar, IL of salt water, pH : 7.2-7.4) for the detection of Flexibacter maritimus

(Pazos et al., 1996).

Sampling

Two series of samplings were done: one in June at the beginning of the experiment and

another in August at the end, to ensure that it was the same pathogen in both cases. Five

sick fish were removed before feeding in the different tanks. These fish were transported

alive to the laboratory and analysed directly upon arrivai to avoid degeneration of tissue

that could cause an uncertain diagnostic. Fish were sacrificed by a blow on the head,

adipose and caudal fins were sectioned and stored in 95% ethanol for future analysis

whereas weight and forklength were measured. Externallesians were the mast prominent at

21

gills, tale or tlank. These lesions were cleaned with 70% ethanol, then incised with a sterile

scalpel. Sowing by scratches in aseptic conditions were made with an inoculation loop on

the different culture medium. Triplicate plates labelled for each charr sampled were

maintained in the dark at room temperature and frequently examined. Three isolations were

realised to obtain a single type ofbacteria. We picked representative isolates of each colony

type observed for characterization and identification. A drop of the suspended culture to be

examined was transferred on a microscope slide with an inoculation loop and was stained

using the 77730 Gram Staining Kit following the manufacturer' s indications (Fluka, Neu-

Ulm, Germany). Representative isolates were removed, placed in aliquot of 1 ml containing

1501-11 of autoclaved water and stocked at -20°C. In order to confirm our preliminary

results, a nested polymerase chain reaction system developed by Cepeda et al. (2003) was

applied to detect Flexibacter maritimus from fish tissue. Clinical isolate of Flexibacter

maritimus (ATCC 43398) provided by Uhland (Faculté de médecine vétérinaire, Université

de Montréal, Canada) was used to test the sensitivity of the nested PCR proto col as

reference strain. The stain was stored in vials at -80°C in Marine agar broth (Difco

Laboratories, USA) with 20% glycerol added. DNA from the representative isolates was

purified by Phenol: Chloroform: Isoamyl (Alcohol 25 : 24: 1 saturated with 10mM Tris, pH

8.0, ImM EDTA) (Sigma-Aldrich, Germany). The reference strain was used as positive

control whereas an Escherichia coli DNA and a tube without any DNA constituted our

negative controls.

22

Primers

According to the protocol described by Cepeda et al. (2003), two universal primers, 20F (5 ' -

AGAGTTTGATCATGGCTCAG-3 ') and 1500R (5 ' -GGTTACCTTGTTACGACTT-3 '),

complementary to conserved regions of most eubacterial 16S rRNA, were used for the first

PCR step. For the second PCR, two specific primers MarI (5 ' -TGTAGCTTGCTACAGATGA-3 ')

and Mar2 (5 ' -AAATACCTACTCGTAGGTACG-3 ') expected to give a product of 400 bp were

used. Nucleotides were synthesized by Biocorp Inc. (Montréal, Canada). The cycling

conditions for PCRI were 30 cycles of denaturation (95°C for 30 seconds), annealing (57°C

for 30 seconds), and extension (72°C for 60 seconds). A preheating step at 95°C for 5

minutes and a final extension step of 5 minutes at 72°C were done. The cycling conditions

for PCR 2 consisted of a preheating step of 94°C for 2 min, followed by 40 cycles at 94°C

for 1 second, 54°C for two seconds and 5 seconds at 72°C, followed by a final extension

step of 72°C for 4 minutes. The PCR products were analysed on a 1 % agarose gel.

2.2.4 Stress exposure

The experiments were conducted from the beginning of June to mid-September 2005 . At

the beginning of June, both temperatures offresh and seawater reached 8°C and, charr aged

of 1 + were gradually transferred to salt water (i/oo per day over 10 days) until salinity

reached 20%0. Ten days later, an antibiotic Onycin 1000 (Vetoquinol, Canada) treatment

was applied to follow regular rearing procedure (tetracycline HCI powder: 75 mg kg-1 body

weight day"1 for 10 days) . Twice a week, salinity was adjusted with a refractometer. Charr

23

were monitored throughout the day, sick fish were removed, and mortalities were recorded

in the morning before the feeding and late afternoon.

Samples ofDNA were obtained from either adipose or caudal fin from sick fish and

preserved in 95% ethanol to constitute a genetic bank. Clinical signs, forklength and weight

were also noted for dead fish. At the beginning of August (1 st and 8th August), two acute

stress were applied in sequence. The water in each tank was drained to three quarters. Fish

stayed in low water depth for a period of 10 minutes before tank was refilled. In mid-

August, salinity was increased to 30%0. The experiment stopped in mid-September, when

ch arr were transferred back to freshwater.

Fish derived from a diverse set of crosses, failed to cluster into "resistant" and

"susceptible" phenotype groups. Based on the difTerent families responses observed, we

determined four scales of disease susceptibility : 1°) unafTected (0 mortality) or very

resistant (less than 10 mortalities ail along the experiment); 2°) very sensitive: affected by

the transfer to 20%0 (more than 10 mortalities); 3°) moderately sensitive: affected by acute

stress (more than 10 mortalities); 4°) slightly sensitive: afTected by the transfer to high

salinity 30%0 (more th an 10 mortalities) .

2.2.5 Statistical analysis

Data on growth performance were gathered into databases and statistical analyses were

performed using STATISTICA 6.0 (Statsoft Inc., France). In order to analyse our two

24

different types of hybrids (intra and interspecific), the 25 families were pooled in two

different groups representing intra- and interspecific crosses. One was composed of

Salvelinus fontinalis : anadromous*anadromous (AA), anadromous*resident (AR),

resident*resident (RR) and the other one of S fontinalis : anadromous*anadromous (AA),

anadromous hybrids S. fontinalis*S alpinus (CF), S alpinus: anadromous* anadromous

(CC). Two-way ANOVAs (a = 0.05) were performed (family, time of sampling) on the

following variables: body weight, fork length and condition factor (100*(weight/length)3).

The normality and homogeneity of variances were checked by Komolgorov-Smirnov and

Levene tests respectively. Weight, length and condition factor data had to be transformed as

log (x) to obtain normality. Wh en there were significant interactions, a posteriori one-way

ANOVAs were realised followed by Tukey HSD post hoc tests. In absence of

heteroscedasticity, pairwise comparisons were realised using Games and Howell's test

(Sokal and Rohlf, 1995).

2.3 ResuUs

2.3.1 Parental genotypes of MHC class II (region j31) aile les

A total of 21 MHC class II (region ~ 1) exon 2 alleles were identified from

broodstock and assigned by an alphabetic letter from a to u (Figure 1). Among the 271

nucleotides and 91 amino acid positions, 79 (29%) nucleotide positions and 41 (45%)

ami no acids residues were variable (Figures 1 and 2).

a b c d e f

9 h i j k 1 m

n o p q r 5

t u

a b c d e f

9 h i j k 1 m n o p q r 5

t u

1

GATGGATATT

91

TCAGATTGAA . A .. G .... . . A • . GC ... . .•• . G • ....

.•• . G ....•

.•• . G .... .

.•• . G .... .

. A .. G .... .

. A .. G .... .

. A .. G .... • ••• • GC ••• • • ... G .•••• . A .. GC ....

. A . • GC ••• •

. A .. GC ••••

... • G ..... ••• • G • • • ••

.... G .• • ..

... . G ...•. • .. . G ...•. • •• • G .•..•

11 TTTGAACAGG · .- • .• . . . A

• . -T . T •••• • . AAT - • • TA · . AAT - .. TA

• . AAT - • • TA • .• CTT .• • A . ..• .. . .. A • ••• •• • • • A . .. - . T .. • A . .. A T - • . TA ... AT - .. TA .• • AT - ••• A • •• AT - ••• A • •• AT - • • • A • • • AT - • • TA .• • AT - •• TA ... . ... . TA .... . ... TA ... - .. . . TA .. . GT - .. TA

10 1

CACATCAGAT G ..• ••••• • T . TG .. • ... A .... ... . . A ... .. . . •• · . A . .... . . A .... ... . . G ........ . G •.. • .... • G • .. ••• .• • T.TG •••• • • T .TG .• • ••• T . TG . . . . . .

T . TG •• •• •• T . TG . T •• • • G . T . .... . . G . T • •••••• G . T • •. • •.. G . T ...... . G . T ... . .. . G . T •••....

2 1

TTGTGAGACA GG . . • • ••••

GGT .. GT .. . GGT . . GT . • •

GGT • • GT ••• CG . . .. C .. . GG • .• •••• •

GG • • •••••• GG .. . G .... GGT . . GT ••.

GGT • • GT • •• GG • • • G ••• • GG • •• G •• •• GG • • • G •••• GGT •• GC • • • GGT • • GC • •• GG • • TG . T •• GGT • • GT • •• AC . • • GATA . GG .. . G • • • .

111 TCAl\.CAGCAC

31

GTGCCGATAC . . . . .. . . T. . •. T . .... .

. . . . . . .. T.

. . .. .... T .

. . . ..... T .

. C ..... . . .

A ••••• • • • •

1 21

TGTGGGG,lI.AG

41

TCCTCAAAGG

13 1

TATGTTGGAT

. T .. ... .. .

. T .. .. . .. .

. T .. ..... .

. T . .... . . .

.T . ..... . .

. T .. . . .. . .

51

ACCTGCATGG

. . ... .. C ..

141

ACACTGAGCA

• •• •• • • • GT • • • •• • •• GT • •• •• • •• GT • •• • • ••• GT . . ... • • . • T . .... •. .. T

. . . .. . ... T

. .. . .... T.

... . .... T.

. .... . .. T .

. .... .. . T .

. ... . ... T .

61

TATAGAGTTT

. GC .. .. . . . . • •• • . • •• G • ••• •• • •• G

• ••• ..... G • ••• ••••. G

. .. .. . .. A .

. .. . . . .. A .

. ... .... A .

.GC .•. • •••

. GC • •• • • • •

15 1

TGGTGTGAAG

• ...... T . C G .•. T ... G . G •• . T ..• • • G .. . T . . . . . G ... T .... . G ... T . ... . G . .. T .. . . . G . . . T . . . . .

. ... ... T . C • • • • • •• T . C

. . . .. . . T . C

71

ATAGACTCTT

1 61

Nl.. TGCAG,lI».

...... A .••

. •. .. • ••• G

• • . •• • • • • G

• • .•.• • •• G

. C .. ..... .

. C .. . .... .

. C .... . .. • • • ..• •• • • G

• • •• •• • • • G

• • • •• • • •• G

. • . • . • ••• G

• • • • • • • •• G

• • • •• •• • • G

• ••• • •• • GG

81

ATGTTTTCAA

. CTAC • •• • •

. CTAC • •• ••

. CT AC •• • ••

. CTAC •. • ••

171 CATGGAACAA

• ..••.• • • G

• • •••.. • . G

T • •• • •• • • G T • ..•.. . • G

N Vl

181 a AGGTTCTGTC b c •. • .... . AG d • . •..... A. e . ....... A . f ........ A . g .. . ..... AG h · . A ..... GG

. A ..•.. GG j .. A ..... GG k • . A .. ---A .

. A .. ---A. m · . A . . ---A. n · . A .. - --A . D .•....•• AG P ... ...•• AG q . .•.•.•• AG r · . AG .... A. s · .AG .••• A. t ... G ..•• AG u ....... . AG

271 a ACAGGTG b c d e f g h

j k 1 m n D

p q r s t u

191 201 211 221 231 241 251 261

---CTGGCTC GAGAGCTAGG GGAGCTGGAG CGTTTCTGTA AGCATAACGC TGATCTCTAC TACAGCACCGT ACTGGATAAG

- -- .... T ..

ATT • ..•.•• ATT ...... . ATT ......•

A •. . . . ...• A ••.•..•.• A .. . •.• ... A •••• • . • •• A ••••.••.• A ........ . A .....••.. A •••.•. • •• A •••••• • •• A ••••••••• A •• •••• .•• A .... • AG.C A . ...• AG.C A . .... . .. .

.C ...... .. • C ....... .

. ....... C .

...... .. C .

. ....... C .

. ... A ... C .

. . . GA . . . C .

...... .. C .

.. ...... C .

...... .. C.

.. .. A ... C .

.. ...... C. A •.• A •.. C. A •.. A ... C . . .. G .. .. C . .. . G .•.. C .

· .T ...... .

· .T ... .. . . ..T ...... .

.. .C.C . .•.

... C .... ..

..T .. . ... .

..T .. ... . . · .T ... C . . . ... C .. .. . .

.... A .. C ..

.. .. A .. C • .

· . C .AA . C . . · . C .. T;A.C .. · . C.AA . C •. .. .. A .• C •• . .. .. .. C .. . .. .... C .• .. ... . . C • . .. C .A ... . . · . C ... . C . . . ...... C .. .. ..... C .. · . C.AA . C •. . .. . A ..... · . C.AA.C •• .. C. A .... . .. C.A .... . · . C.A . . G.A . ...... C ..

• .. ... G .. A • • ..... G .. A. . .. ... G.---. • •..• G .. • •

. .. ... G.---

. . .... G. ---

. . .... G .. A.

.. • • •• G •. A • • ..... G .. A . . .•.••• A . •• . ••.••• A .•. • •• G •• G •. A. . •• G •• G •.• .

. ••••• G •. A .

.. •••• G •. ••

.. . ... G. --• ••••• • A .• . .•••••• A . • . . . . • •• G •.• .

. .. G •• G •.•.

• ..... C ...

.. •... C •••

Figure 1. Exon 2 sequences of the MHC class IIJ31 alleles in Salvelinus alpinus and Salvelinus fontinalis . Dots indicate identity with the top sequence.

IV 0\

j ". "

j R

DG-'V ftQ'!::~l~:~ ~,~.~~.R.,.fo,~

~ , , .1~};Rl<! v~' .. .. , , • ln~'F!.L~{c .. # , .1.(! .. ! (

.. ,P..tiR , (}1,'·iR

" ,. '{c~#~. ,Y8.RIN. . Yri.Rl}/ ~

:- " +f.t ,... , ( , ( t . :":.( ::z •.

, ( , • "i~~~L~. , .: ) • Ï~'?tLA.

)~~,VG

) «, .Zfu"i .: ::~

. C'f~~, G,

.. , .. D .. , \ , .. ' ~ ~ . .. , .. ..... ~ , , , ...

n"SI .. ~ 'Q , v'

D,-S! .. ,.Q, .... ~

DI. -. ,'Q, .. -D,y, . ;(: ...

E" :: ~ "Qt~~ ~, ~r;~ .. Q~~;'; ~ ~ tE • .. Q: ~ ... , " ,E; •• " ..

C~~tS S~;)1.H·:; ";'

" + <Co ~

~ , .... ''f , ~ , "x,

Q .... ~S:~l~"'Y~ .:,,~ .::,"":,( V )e'~""''''~~' ''' ' of.~

" <t . .. .. "!J ~ ... '::"'! ,

. ' k '" IHDSTifl!

• , L" .. ,.,

~t,,~ + ~ .. ~,. .. t, ~~, . .. # ,

+ • , • 1> • , • , •

,

, "t),' .. c , c" , 't, ' .. c , • ,

~. , <( ,. t ·

l4 ** .. * ,t

DLY~·5T~~·~i{T

· !.li. ,,~:L , . , , !~L "A! • < > ,

~}~iL~~~ " , .. ~;Nn; ~ ... _ .... _ .... -,IiL ,------• .,li. ,J;!.,. , .' • .fi. ,AT.,., " iL ,J'i.!,." ll.! , . ,g, . ,. , ll. . .fi . ,g, . , . , • ,lfiGJ'i.L . "

,I~) .. AC ~ t'~

~l~r'L , ------);,1,. ,g,. , ., !I#" "H ... t ~ " ~!!. t ~ Â~., t )

H

QIEfiTRmSr ~:VtD~ .. .. ,+.

.. ~J~Q~ . .. # ... , .

,\~,n .. t .. , ••

k-'1 t D .... ~ ~ . kVcD., c, •. .

c "'ll . "'''l'/, c , + •

~,r.,te , ( .• :t'f.A., TF., <{ ••

~,"'''l'~~,., +.

,:],D, c, «

,:;, D~ c , <{

e"':.{' .. ::c , « , i ' ~

,"!.J,!::, c, ( • c

• Y:i ~ J. , . , ~ . .

,t

t,:gifNGïTE: H,

.. F .. ~r " " '(

<t- .. ! ~t , " ~ .. <t

':' '/ of. .... c • . · , Y . , . ~ ~.,~

~ .. 'i ~ , , .. 1 ,' +' y, . ,' , ,'L

· · Y. .. .1, <>

<t- .. L .. , , .. 1 ., Y. , ." y

· · le · " · ~t · · x. · · r .. Y. , '« ~ ... ~

· ~F . · · if · .F. · · if

. ' c , , ~ o' ~ ~ , t

" .. ~~,. #K c ~ , ,

, .. tR" , i;". ,

,. L# , .' .. ;~ .. , ,. . L;# , 0' <t- ~ .. , ,

•. L, t 1> ~ ~ ( , ,

... L, <{ > " , ~ , «

,t.".· .fo, ('" .. ( , ~ ~ 0' ~( ,,R

c, ( ~, Ac,R

, ( , ,. ~. ~;, (p., , e , ... V, e ~t

, , Of, .. + • ) • ,

, ~{, • + • , <{ ,

... .. • , .. ~ ,x:. <( ) ..

. , ï ) . ... ) ( ) <t- . ) , t ·,. , ,1

27

Figure 2, Amino-acid sequences for MHC class 11/31 alleles ln Salvelinus alpinus and

Salvelinus fontinalis. Codons involved in antigen binding in human (Langefors et al. , 2001 )

are denoted by asterisks.

28

From the 25 positions of the antigen-binding sites (noted by asterisks), 19 (76%) were

variable (Figure 2). AlI alleles encoded distinct amino-acid sequences, at the exception of

alleles i and j , which differed from one nucleotide. Five specific alleles (a, c, g, q and t)

were found only in anadromous Salvelinus alpinus. It also appeared that six alleles (e, f, k,

l, 0 and u) and four alleles (h, j, p and r) were respectively specific to anadromous and

resident Salvelinus fontinalis. Six others (b, d, i, m, n, and s) were shared between

anadromous Arctic and anadromous or resident brook charr. Three (P, l , L) of the four

alleles (P, H, l , L) identified by d'Autray Tarte (2004) in Salvelinus fontinalis (Laval

strain), were identical to ours, and corresponded respectively to alleles h, d and m. SSCP

analysis revealed that sorne parents present the same sequence as their DNA strands assume

the same shape, and consequently similar gel mobility (Sunnucks et al., 2000) (Figure 3).

Looking at the allele frequencies, it appeared that seven alleles (i, c, d, s, p, n, g) occured

more frequently than others (over 5) and that six alleles (e, f, k, 0 , rand u) were only found

in one individual (Table 2). Only three dams and three sires were homozygotes, the 37

others were heterozygotes (Table 3). From the parental genotypes, an estimation of possible

allelic combinations that should be found in progeny was made (Table 3). There was no

similarity in allelic combinations among the different families .

2.3.2 Identification of pathogens

Samples obtained from lesions on ten moribund fish were first characterized by

efficient selective media. After incubation in dark at room temperature for 2 to 3 days,

29

Figure 3 : Silver-stained SSCP gel of the MHC class II pl from Salvelinus fontinalis and Salvelinus alpinus. Lanes: l , molecular weight marker; 2, anadromous Salvelinus fontinalis sire BC046; 3, anadromous Salvelinus fontinalis sire BC045; 4, anadromous Salvelinus fontinalis dam 1151 ; 5, anadromous Salvelinus fontinalis dam 1152; 6, anadromous Salvelinus fontinalis sire BC044; 7, anadromous Salvelinus fontinalis dam 1150 not used for the experiment; 8, anadromous Salvelinus fontinalis dam J056; 9, anadromous Salvelinus fontinalis dam 1157; 10, resident Salvelinus fontinalis sire V002; 11 , anadromous Salvelinus alpinus sire BC079; 12, resident Salvelinus fontinalis dam ROOO res; 13, resident Salvelinusfontinalis dam R006 not used for the experiment (refer Table 1).

Allele Frequence

a 3 b 2 c 8 d 8 e 1 f 1 g S h 2

10 J 2 k 1 1 2 m 3 n 6 0 1 P 7 q 3 r 1 s 8 t 4 u 1

Dams

BC070 J159

BC061, BC066, BC069, BC054, BC053, BC070 ROOO, R049, ROS8, BC040, BC039

R053 BC055, BC056, BC069, BC053

J153,J155 J157, J056, J151

JO 62 ROOS

ROOS, ROOO J159, BC040

ROS8, J062, J075, B014, BC039

J153, J155, J075, B014 BC061, BC056

J157 J056, J152

BC055 R049

Sires

BC068, BC060 V002

BC060, BC062 V060, VOOO, VOOI

VOOI

BC065

VOOS, BC045, BC043, J152, BC047, BC046, BC044 BC057

V060 V002 V062

BC043, BC057, BC047 BC079

VOOS, BC041, BC045, J151, BC046, BC044 BC065, BC062, BC068

Table 2 : Allele frequencies from broodstock. Bold character indicates anadromous Salvelinus alpinus individuals, italic indicates anadromous Salvelinus fontinalis individuals and normal character indicates resident Salvelinus fontinalis individuals. Five alleles (a, c, g, q and t) are specifie to anadromous Salvelinus alpinus, six alleles (e, f, k, 1, 0, u) are specifie to resident Salvelinus fontinalis, and four alleles (h, j, p, r) are specifie to anadromous Salvelinus fontinalis.

w o

31

Cross Family Dam Sire Dam Sire Expected progeny genotypes genotype genotype

AA Al J062 BC041 nJ sh ns, nh, js, jh A2 J075 BC045 np SI ns, m, ps, IP A3 J151 BC043 SI IP SI, sp, 11, IP A4 J152 BC044 SI SI SS, SI, 11

A6 B014 BC046 np SI ns, m, ps, Ip AR AR1 J157 V002 fi nb rn, rb, ni, bi

AR2 J153 VOOO hp dd hd, dp AR3 J056 V001 SI de sd, se, di, ie AR4 J159 V060 mb md mm, md, mb, bd AR5 J155 V005 hp SI hi, sh, ip, sp

RR R2 R005 V005 kl SI li, ls, ks, ki R3 ROOO V002 dl nb nd, ni, bd, bl R6 R053 V062 fI 00 fo R7 R049 V060 ud md um, dd, ud, md R8 R058 VOOO nd dd nd, dd

CF CF1 BC054 BC046 cc SI CS, CI CF2 BC053 BC041 cg sh CS, ch, gs, gh CF5 BC040 BC057 md JP mj , mp, dj , dp CF6 BC039 BC047 nd Ip ni, np, di, dp CF7 BC070 BC045 . .

ca SI CS, CI, as, ai

CC C2 BC061 BC060 cq ca CC, ca, cq, qa C4 BC066 BC065 cc gt cg, ct C5 BC055 BC062 gt ct cg, gt, ct, tt C6 BC056 BC068 gq at ga, gt, qa, qt C9 BC069 BC079 cg qq cq, gq

Table 3. Parental and expected progeny genotypes for Salvelinus fontinalis and Salvelinus alpinus.

32

white-yellow colonies, appeared on AOA and FMM, whereas nothing was observed on

TCBS at the exception of the triplicate plates from one fish. Nevertheless, colonies did not

exhibit the typical Vibrio sp morphology characteristics, which are flat and large yellow

colonies (Difco and BBL Manual, 2003).

Further analysis showed the presence of long and thin gram-negative rods. The

presence of Flexibacter maritimus was presumed by the appearance of colonies

morphology, pale-yellow, flat with uneven edges, as described by Toranzo et al. (2005) .

This preliminary diagnosis of marine flexibacteriosis was supported by the use of specific

molecular DNA-based methods. The nested PCR amplification generated a 400 bp product

for the reference strain Flexibacter maritimus (ATCC 43398), whereas amplification

products were not obtained from our two negative controls. Each batch of samples was

amplified with positive and negative controls and for both series of samplings, one single

band at 400 bp length was observed (Figure 4). Flexibacter maritimus was isolated and

identified as the causative agent of opportunist lesions in aIl cases.

2.3.3 Stress exposure

Fish condition during the growing season

Ali families were transferred to salt water at the beginning of June. Weight and

length were measured in April, July and September to follow their growth performance

during summer. From these data, condition factors, which indicate fish condition, were

33

calculated. Familial differences were found for each sampling period, families did not keep

the same profile in time. This led us to pool and analyse them according to two groups:

intraspecific (AA, AR, RR), and interspecific (M CF, CC) crosses. Significant

interactions among intraspecific families and sampling time were present. The analysis of

variance results are summarized in (Table 4).

34

1 2 3 4 5 6 7 8

Figure 4. Nested PCR products obtained from sick fish. Lanes: 1, molecular weight marker; 2-3, negative controls: one without DNA and one with Escherichia coli DNA; 4, positive control : reference strain Flexibacter maritimus, clinical isolate provided by Uhland (ATCC 43398); 5, molecular weight marker; 6-7, two examples from each batch of samples to show the presence of a distinct single band at 400 bp but it was aiso the case for ail others samples; 8, molecular weight marker.

35

GrOUE Variables Effect df F E-Ievel Intraspecific Weigth Family 14 62.4 <0.001

Sampling 2 1768.1 <0.001 Family* Sampling 28 2.4 <0.001

Length Family 14 44.4 <0.001 Sampling 2 990.0 <0.001 Family*Sampling 28 1.3 <0.001

Condition factor Family 14 63 .8 <0.001 Sampling 2 582.0 <0.001 Family* SamEling 28 5.6 <0.001

Interspecific Weigth Family 14 43.4 <0.001 Sampling 2 1307.3 <0.001 Family* Sampling 28 1.9 <0.001

Length Family 14 67.0 <0.001 Sampling 2 1517.4 <0.001 Family* Sampling 28 1.8 0.125

Condition factor Family 14 45 .1 <0.001 Sampling 2 632.3 <0.001 Family* Sampling 28 8.8 <0.001

Table 4. Two-way ANOV A results. Comparisons among intraspecific Salvelinus fontinalis (AA, AR, RR) and among interspecific Salvelinus alpinus - Salvelinus fontinalis progenies (AA, CF, CC) and among the three sampling periods (April, before the transfer in salt water; July, during the transfer; and September, just after the transfer) (refer to Table 1).

36

In spite of these differences, we could notice that during the whole summer, the family of

smallest size was A2 and the largest one, R6 (Figures 5-6). However, the se families did not

have the lowest and the highest condition factors (Table 4). Condition factors were

particularly variable in April, ranging from 0.749 (CF2) to 1.076 (R8). Salvelinus fontinalis

resident families (principaIly families R8 and R2) tended to have the highest condition

factor aIl along the summer. In April, anadromous families had the lowest condition factors

and were among the smallest and the largest families . AlI families grew weIl during

summer time, but the differences observed in April were generally maintained throughout

the season. Conditions factors were generally higher in September, at the exception of

family AR2, which had a higher one in July, period corresponding to a remarkable growth

performance.

Considering the interspecific comparisons, families differed significantly in weight

and condition factor but not in length between sampling time (Table 4). As with

intraspecific families, in April, anadromous families were among the smallest and the

largest size families, but in this case, their condition factors were among the highest.

During the whole summer, the smaIlest families were CF5 and A2, whereas the largest

ones, A4 and CF6 (Figure 7-8). In April, conditions factors were low, majority was inferior

to 0.9, however in September, aIl families had condition factors over 1.0 (Table 5). AlI

families grew weIl during summer time, but C9, C5, CF7, and C6 grew considerably from

July to September (Figure 8 and Table 5). In April, brook charr families had the highest

37

condition factors, but the situation changed after seawater transfer. In September, Arctic

charr had higher condition factors than brook charr.

180

160

140

120

-C) - 100 -.c C)

'Qi 80 3:

60

40

20

0

cO'

April July

<0

Ol CT

Ol 0 CT Ol

cO' :::T

CT

Ol 8. §-Q.CT (t) ~ ::t" CDOcO'~ aft

cO' Ol CT

8. Ol

CD s:

<0 :::T

September

CD cO'

:::T

Ol CD CT

cO'8.

m.

Figure 5. Fish weigth of the intraspecific Salve lin us fontinalis families (AA, AR, RR) during summer time. Mean ± standard deviation. Significant interactions were found for each time of sampling, results of a posteriori test are represented by a letter: abcdefg.

w 00

30

251 cO' <0

<0

.:::- "T

20 -E (.) -J: 15 -C) C CI) -J

10

5 ~1~1l;1~ 16;1~1~ ~lal~I~I~ ~1~ 1l; 1~16;1~1~1~1~1~

...... I\JW"c:..01

• o l ' . ,

April July September

Figure 6. Fish length of the intraspecific Salvelinus fontinalis families (AA, AR, RR) during summer time. Mean ± standard deviation. Significant interactions were found for each time of sampling, results of a posteriori test are represented by a letter: abcdefg.

w \0

40

Group Farnily Condition factor AEril July SeEternber

Intraspecific Al 0 .897 ±O.064 (50)a 1.003 ±O.053 (30) alx: 1.035 ±O.062 (50) ac

A2 0.926 ±O.047 (50) ab 1.044 ±O.049 (30) bode 1.118 ±0.162 (50) bodf

A3 0.981 ±O.069 (50)bod 1.084 ±O.043 (30) ef 1.129 ±O.078 (50) bdef

A4 0.956 ±O.049 (50) b 0.970 ±O.055 (30) a 1.029 ±0.197 (50) ab

A6 0.908 ±O.041 (50)a 0.960 ±0.083 (30) b 1.006 ±0.056 (50) a

ARI 0.923 ±O.073 (50) ab 1.051 ±O.046 (30)cde 1.122 ±0.074 (50) bde

AR2 1.027 ±O.063 (50) de 1.138 ±O.069 (30) fg 1.115 ±0.076 (50) bde AR3 0.961 ±0.057 (50) lx: 1.037 ±O.055 (30) abode 1.126 ±0.073 (50) abodef

AR4 0.897 ±0.049 (50)a 1.028 ±O.038 (30) abod 1.039 ±0.042 (50) ac

AR5 1.011 ±0.060 (50)cde 1.102 ±0.063 (30) efg 1.123 ±0.074 (50) bde

R2 0.987 ±O.115 (50) abode 1.175 ±0.055 (30)g 1.200 ±0.069 (50) f

R3 1.044 ±0.082 (50) de 1.109 ±0.074 (30) efg 1.172 ±0.053 (50)"f

R6 1.056 ±0.084 (50) de 1.100 ±0.046 (30) ef 1.130 ±0.142 (50) bde

R7 0 .950 ±0.075 (50)alx: 1.079 ±0.055 (30) def 1.108 ±0.071 (50)bd

R8 1.076 ±0.083 (50) e 1.172 ±O.059 (30)g 1.194 ±0.058 (50)f

Interspecific Al 0 .897 ±O.064 (50)a 1.003 ±O.053 (30) alx: 1.035 ±0.062 (50) ac

A2 0.926 ±O.047 (50) ab 1.044 ±0.049 (30) bode 1.118 ±0.162 (50) bodf

A3 0.981 ±O.069 (50)bod 1.084 ±O.043 (30) ef 1.129 ±0.078 (50) bdef

A4 0.956 ±O.049 (50) b 0.970 ±O.055 (30) a 1.029 ±O. 197 (50) ab

A6 0.908 ±0.041 (50) a 0.960±0.083 (30)b 1.006 ±0.056 (50) a

CFI 0.938 ±O.088 (50) de 1.063 ±O. 134 (30) bode 1.088 ±0.089 (50) bod

CF2 0.749 ±0.058 (50) a 0.818 ±0.084 (30)a 1.003 ±0.059 (50) a

CF5 0.790 ±0.092 (50) ab 0 .907 ±O. 107 (30) ab 1.104 ±0.090 (50) bod

CF6 0.988 ±O. 103 (50) e 1.129 ±O. 138 (30) e 1.147 ±0.150 (50) bod

CF7 0.892 ±0.071 (50) cd 1.002 ±0.124 (30) bode 1.045 ±0.161 (50)ab

C2 0.928 ±0.115 (50) de 0.991 ±0.074 (30)bod 1.090 ±0.080 (50) bod

C4 0.811 ±0.122 (50)alx: 0.937 ±0.082 (30) bd 1.078 ±O.068 (50) lx:

C5 0.963 ±0.104 (50) de 1.005 ±0.077 (30) bode 1.163 ±0.076 (50) d

C6 0 .878 ±0.151 (50)lx:e 0.898 ±0.144 (30) abod 1.141 ±O.097 (50) cd

C9 0 .963 ±0.125 (50) de 1.051 ±0.096 (30) cde 1.175 ±0.096 (50) d

Table 5. Condition factor of intraspecific Salvelinus fontinalis (AA, AR, RR) and interspecific Salvelinus alpinus - Salvelinus fontinalis (AA, CF, CC) farnilies during surnrner tirne (refer to Table 1). Mean ± standard deviation. Significant interactions were found for each tirne, results of a posteriori test are represented by a letter: abcdef.

180

160

140

120

-C) - 100 ... .c C)

'Qi 80 3:

60

40

20

o 1 -r IIV, VI! V.JI .....

April July September

Figure 7. Fish weigth of the interspecific Salvelinus alpinus* Salvelinus fontinalis families (AA, CF, CC) during summer time. Mean ± standard deviation. Significant interactions were found for each time of sampling, results of a posteriori test are represented by a letter: abcdef.

~ ........

30

25

20

Ê (J -J:;15 ë, c: Q)

...J

10

5 ~ K'i ~ i:

CF5 A2 A3 A 1 CF1 C9 C4 CF? CF2 CS C2 A6 CF6 C6 A4

5; () "'Tl ....

() "'Tl N

() "'Tl (J1

() "'Tl (j)

() "'Tl -..j

O+I~~~~~~~~~~~~~~~~~~,-~~~~~~~~

September

Figure 8. Fish length of the interspecific Salvelinus alpinus* Salvelinus fontinalis families (AA, CF, CC) in September. Mean ± standard deviation. There was no significant difference between time of sampling. Interfamilial differences for September are represented here but the same profile has been found in April and July. Results of pairwise comparisons using Games and Howell ' s test are presented here for fish length of the interspecific families.

~ N

43

Disease susceptibility

Disease susceptibility varied tremendously among families . The most resistant ones, in

which no mortality occurred during summer time were S. fontinalis anadromous (A4, A6),

resident (R7) and interspecific hybrids S. fontinalis* S. alpinus (CF6, CF7) (Figures 9, 10,

12). In sorne families, apparition of lesions and loss of appetite occurred two weeks after

transfer to 20%0. Lesions were superficial when mortalities occurred early, but developed

into eroded ulcers when mortalities took place later during the experiment. The most

prominent clinical signs in both species were erosive lesions on the head, dorsal and lateral

sides of fish. Many cases of fin or tail rot, jaw erosion or different combinations of these

were reported. It was not rare to find dead fish without visible lesions.

These families in which mortalities occurred early in summer were considered as

the less resistant to opportunistic infections. These families were S. fontinalis anadromous

(Al , A2, A3) and resident (R8, R2, R3) (Figures 9-10). Following exposure to acute stress,

few mortalities were recorded (less than 3) in S. fontinalis resident (R3 , R8), intraspeficic

hybrids (AR 1 , ARS), interspecific hybrids (CF2, CFS), and S. alpinus (C4, C6) (Figures lO-

B). On the other hand, important mortalities occurred upon transfer on individuals to high

salinity conditions. Susceptible families after transfer to 30%0 were S. fontinalis resident

(R8), intaspecific hybrids (ARS, AR3, AR1 , AR2), and S. alpinus (C4, C2, C9) (Figures 10;

Il ; 13). Sorne families, S. fontinalis resident (R6), intraspecific hybrid (AR4), interspecific

hybrids (CFI , CF2, CFS), and S. alpinus (CS, C6), were considered very resistant due to

few mortalities recorded during the entire summer (less than 10) (Figures 10-13).

40

35

30

u III 20 u Q.)

> iS 15 :i E :::> ü 10

5

o

-+- family A3 -+- family A 1 -+- family A2

.. family A4 ~' family A6

u c: Q.)

ë Q.)

E 1iî ~ ü '5 :n :g al

·5 L---~----~--~----~--~----~--~----~----~--~-----o 10 20 30 40 50 60 70 80 90 100 110

number of days after transfer

44

Figure 9. Cumulative mortalities in anadromous Salvelinus fontinalis families (AA) during summer time. Dotted arrows indicate that no mortality occurred in families A4 and A6.

40

35

30

.c 25 fi)

--= u III 20 u Q.)

> iS 15 :i E :::> ü 10

5

o

-- familyR2 ""* family R3

-- family R6 family R8

.- family R7

-::, N

:u c;; c:

~ t

u c: Q.)

ë Q.)

E ... 1iî a-~ t Ü

'5 :u :n c;; / :g u c: al c: al N

~ + .. t

·5 L---~----~--~----~--~----~--~----~--~----~--~ o 10 20 30 40 50 60 70 80 90 100 110


Figure 10. Cumulative mortalities in resident Salvelinus fontinalis families (RR) during summer time. Dotted arrows indicate that no mortality occurred in fam ily R7.

40

35

30

oC 25 II!

;;::: u Xl 20 u <Il

.~ li 15 3 E ::J U 10

5

o

-- family AR1 -- familyAR2

family AR3 -- familyAR4 -+- family AR5

-5 ~--~----~--~----~--~----~--~----~--~~--~--~ o 10 20 30 40 50 60 70 80 90 100 11()

number of days atter t ransfer

45

Figure I l . Cumulative mortalities in anadomous-resident Sa/velinus fontinalis fami lies (AR) during summer time

40

35 family CF1

-s-- family CF2 family CF5

30 .~ family CF6 ~ family CF7

oC 25

E u u -c :Il 20 <Il 0 U C M <Il <Il II! :u > E il

II! (;:; 15 li II! <Il

3 ~ II! II c <Il ~ E - II u ::J U

u b :g c 10 N "0 N t

Qi :.0 ~ t r 'E (;:; CIl

~ 5 c

t ~ t --0

-5 0 10 20 30 40 50 60 70 80 90 100 110

number of days after transfert

Figure 12. Cumulative mortalities in anadromous Sa/velinus a/pinus * Salvelinus fontinalis families (CF) during summer time. Dotted arrows indicate that no mortality occurred in families CF6 and CF7.

46

40

35 ___ family C2

--- family C4 --- family C5

30 family C6

--- family C9

.c. 25 0 II) ('") '+= "0 "0 ~ <Il 20 c ;;; Ol) Ol) "0 ë c Ol) Ol) II) ~ > E (f) =a 15 1iî II) Ol)

"5 ~ II) t;

E Ol)

::l - U t; "0 U 0 c

10 N =a (;j N

~ il ~ ;;; <Il

5 c ~

0 t

-5 0 10 20 30 40 50 60 70 80 90 100 110


Figure 13 . Cumulative mortalities in anadromous Salve lin us alpinus families (CC) during summer time.

47

2.4 Discussion

Based on an efficient specific medium selection, we were able to make an initial

diagnosis of the pathogen affecting fish. FMM was chosen due to its ability to allow better

growth of Tenacibaculum maritimum in comparison to the growth of heterotrophic

halophilic bacteria, such as Vibrio, Pseudomonas, and Aeromanas species, which usually

overgrow the plates (Avendaîi.o-Herrera et al., 2005). Results were then confirmed through

the nested PCR protocol of Cepeda et al. (2003) considered as the most adequate for an

accurate detection of Tenacibaculum maritimum in diagnostic pathology as weil as in

epidemiological studies of gliding bacterial disease in marine fish (Avendaîi.o-Herrera et al. ,

2004).

One of the major problems that may be encountered is to distinguish Tenacibaculum

from other phylogenetically and phenotypically similar species, particularly those of the

genera Flavobacterium and Cytophaga (Toranzo et al., 2005). Formerly, many water

bacteria belonging to Cytophaga and Cytophaga-like bacteria were classified by mistake as

myxobacteria. In fact, until recently, those organisms belonged to the Cytophaga-

Flavobacterium-Bacteroides phylum (Reichenbach, 1999). The heterogeneity of the genera

Cytophaga, Flexibacter, and Flavobacterium has caused considerable confusion in

differentiating the se bacteria (Toranzo et al., 2005). Suzuki et al. (2001) reclassified

Flexibacter maritimus into the genus Tenacibaculum belonging to Flavobacteriaceae

48

family . The application of PCR was a great benefit for the characterization of specifie

pathogens.

The filamentous orgamsm Tenacibaculum maritimum (formerly, Cytophaga

marina, Flexibacter marinus and Flexibacter maritimus) is the causative agent of marine

flexibacteriosis, an important disease widely distributed in cultured marine fishes (Toranzo

et al. , 2005). This etiological agent has the potential to cause severe mortalities in several

fish species reared in seawater around the world (Avendano-Herrera et al. , 2005). It was

first identified in Japan in 1979 (Bernadet, 1997) and was reported as a salt-water requiring

organism producing infections characterized by eroded mouth and fins and skin lesions

(Wakabayashi et al., 1986). Young fish suffer a more severe form of the disease than

juveniles (Margarinos et al., 1995). The disease is influenced by a multiplicity of

environmental (stress) and host-related factors (skin surface condition) and could be more

severe if temperatures are above 15°C (Margarinos et al., 1995). Until recently, no vaccines

were available to prevent the disease (Bernadet, 1997), except one for turbot. However, it is

not effective in other marine fish (Santos et al., 1999).

One of our main objective was to verify if differential familial genetic resistance to

this opportunist pathogen in seawater exist. Our results supported this hypothesis as disease

susceptibility varied tremendously among families . Even though, it appeared that the

interspecific hybrids S. alpinus*S. fontinalis (CF) were the most resistant ail along the

experiment. On the other hand, anadromous and resident S. fontinalis were the less resistant

49

to opportunistic infections. However, these families were afTected early in summer but

there was no mortality following exposure to acute stress neither to high salinity conditions.

Furthermore, sorne of the anadromous and resident Salvelinus fontinalis families were

highly or very resistant, showing no or very few mortalities during the whole experiment.

This observation suggested that fish with susceptible phenotype were eliminated from the

beginning of the experiment, and that only the most resistant ones remained.

In general, growth performance was important in ail families in seawater, and

especially in S. alpinus families. Furthermore, in both groups intraspecific (AA, AR, RR)

and interspecific (AA, CF, CC), condition factors were higher and differences reduced in

September, which indicates that at the end of the experiment, ail families were in good

condition. Our results showed that in September, resident S. fontinalis for intraspecific

families and S. Alpinus - S. fontinalis for interspecific families were in better condition than

others. The fact that anadromous S. fontinalis had lower condition factors than resident

throughout the whole summer cou Id be explained by their physiology. Anadromous S.

fontinalis are found to be more streamlined (narrower and shallower bodies) than resident

brook trout (Morinville and Rasmussen, 2003), our data seemed to confirm it.

Families that performed better (A4, A6, CF6, and R6) were highly resistant, and the

smallest families (Al, A2 and A3) were the less resistant ones. However, these

observations should be interpreted with caution because the presence of correlations

between growth performance and disease resistance was not tested. Furthermore, highly

50

resistant families (CF7 and R7), were not among those with largest individuals but were

rather intermediate in size. Afterwards, it will be interesting to calculate dead fish condition

factors in order to compare them with condition factors of surviving fish. If significant

genetic variation in disease resistance is found as it has been the case in many studies, this

will suggest the possibility of selection to enhance resistance to opportunistic infections

(Chevassus and Dorson, 1990; Fjalestad et al., 1993; Wiegertjes et al. , 1996; Cipriano et

al., 2002; Henryon et al., 2002; Roed et al., 2002). According to these studies, in both

cases, inter-specific and intra-specific differences in susceptibility of fish to diseases were

obtained (Cipriano et al. , 2002). However, an important problem in studying susceptibility

in fish populations is to identify and use measurable traits correlating with disease

resistance (Sahoo et al. , 2004). The present study could help in developing strategies for the

selection of marine flexibacteriosis-resistant strains of fish in genetic selection programs.

We isolated and characterized the parental class II ~ 1 alleles in brook and Arctic

broodstocks. Alignment of the 21 alleles' sequences revealed that most of the variability

(76%) resides in the peptide-binding sites. Much of the variation found in the charr

sequences corresponds to polymorphic sites of the ~ 1 domain in higher vertebrates. As

Langefors et al. (2001) noted, this indicates that the polymorphic sites in the fish alleles

differed only slightly from those in human alleles. The high variability of exon 2 was in

accordance with observations made on mammalian class II~ genes (Ono et al., 1993). It is

interesting to note that sorne alleles only differed by a single (d and f; n and m; h and i) or

two nucleotides (d and e; i and j). The fact that most of them encoded for different amino-

51

acid sequences (at the exception of alleles h and i) confirmed the high variability of exon 2.

In their study, Langefors et al., (2001) also found two different alleles that differed by a

single nucleotide. They concluded that one of the se two alleles could be ' an example of a

new allele that has been arisen and been maintained through frequency-dependent selection

due to its association with resistance to furonculosis' (Langefors et al. , 2001).

Specific alleles to anadromous Arctic charr, anadromous and resident brook charr

were found, as weil as alleles being shared between both species. Miller and Withler (1996)

indicated that comparison of Mlle exons within and between closely related species often

reveals the existence of shared allelic lineages, which indicates that the formation of alleles

predated speciation.

The estimation of allelic combinations was realized from the parental genotype. The

progeny inherits two genes, one from each parent, so the progeny proportion is Y4 for each

allele. Nevertheless, future analysis for genotype identification of offspring will be planned

and will confirm the allelic combinations. Based on the high level of heterozygosity from

the parental generation, an important range of allelic combinations is expected to occur in

the progeny. These data will allow to evaluate if progeny survival to infection was

associated with specific alleles. In addition, paternal half-sib crosses will allow us to study

the maternaI effect and to determine if genetic differences exist between resistant and

susceptible individuals.

52

DISCUSSION GÉNÉRALE

Le cadre général de cette étude dépasse un projet de maîtrise car au départ, il avait

été conçu comme un projet de doctorat. La suite du projet sera par conséquent finalisée par

d' autres que moi. Dans le cadre de ma maîtrise, j'ai établi le répertoire des allèles du CMH

classe np 1 chez les géniteurs, identifié l' agent pathogène Flexibacter maritimus

responsable d' infections opportunistes chez les ombles en milieu marin, noté des

différences interfamiliales de résistance lors du transfert en eau de mer et suggéré des pistes

d' analyses pour la poursuite du projet.

Les croisements intra et interspécifiques ont été élaborés pour réunir différentes

combinaisons d 'allèles du CMH classe np 1. Les différences interfamiliales de

résistance/susceptibilité, la présence de 21 allèles différents, et le fait que la majorité des

parents soient hétérozygotes assurent qu'une importante gamme de combinaisons alléliques

sera retrouvée. Une estimation des combinaisons alléliques possibles sachant qu'un

géniteur ne transmet que la moitié de son génome à la descendance (un gène parmi les deux

qu ' il possède) a été réalisée. Les analyses à venir sont tout d' abord de déterminer le

génotype de la progéniture permettant ainsi de confirmer l'estimation allélique établie. En

effet, à partir des prélèvements effectués à la fois chez tous les poissons infectés et chez

cinquante individus pour les familles résistantes, il sera possible de vérifier s' il existe une

disproportion par rapport à l' estimation attendue. Ces résultats couplés avec ceux obtenus

chez les géniteurs, permettront d' établir si les différences de résistance sont corrélées à des

53

différences alléliques au niveau de pl . Il sera alors possible de déterminer si certaines

combinaisons alléliques ou si un allèle spécifique mènent à une plus grande résistance. Si

une variation génétique significative est trouvée, l'identification des allèles conférant une

résistance accrue aux infections permettra d'optimiser la production et de mettre en place

un programme de sélection. Les reproducteurs ayant les meilleures valeurs phénotypiques

pour le caractère recherché, meilleure résistance, pourront être sélectionnés.

Bien que le domaine pl soit la région la plus polymorphique, une étude similaire

concernant le domaine a 1 pourrait être envisagée car cette région est également associée à

la présentation de l' antigène. Chez les poissons, le CMH de classe lIa attire mOInS

l' attention que le classe IIp du fait que chez les mammifères, il est beaucoup mOInS

polymorphique (Marsh et al. , 2000). Cependant, Stet et al. , (2002) ont observé un nombre

comparable d' allèles du CMH de classe lIa que du CMH de classe lIa chez le saumon

atlantique, démontrant ainsi un niveau de polymorphisme de la chaîne a comparable à la

chaîne p.

Les méthodes habituelles de génétique quantitative, pour lesquelles on applique les

lois de Mendel aux caractères présentant une variation discontinue, ne s'appliquent pas

dans notre cas (Falconer and Mackay, 1996). En effet, pour la susceptibilité aux maladies,

on retrouve deux classes phénotypiques : affectés ou non-affectés, mais il n'y a pas

d' intermédiaires. L'infection n'a pas été déclenchée volontairement contrairement à la

plupart des études qui utilisent ainsi le temps de survie comme variable principale. D' après

54

la littérature, la seule analyse envisageable serait d'utiliser un modèle pour lequel les

individus sont affectés s' ils dépassent un certain seuil de susceptibilité (Yang et al., 1998).

Ce modèle : « the liability/threshold model» a été développé par Falconer (1965) pour

traiter les données de « caractères seuils » présentant des distributions discontinues dans le

cas de susceptibilité aux maladies. Selon lui, ces caractères peuvent être analysés de la

même façon que des caractères continus s' ils sont considérés comme exhibant des valeurs

seuils entre lesquelles on observe une discontinuité. Il suggère l'utilisation d'une variable

appelée « liability » qui ne peut être mesurée mais dont la valeur moyenne au sein d 'un

groupe peut être déterminée à partir de l' incidence d'apparition d 'un caractère (ex :

l' apparition d'une maladie). Le point sur l'échelle de « liability » où tous les individus sont

affectés et au-dessous duquel tout le monde est « normal» est appelé un seuil.

Un exemple a été réalisé pour la famille R8 (Annexe 1). Quatre classes

phénotypiques ont été définies : non-affectés, affectés par le transfert à 20%0, affectés par

les stress aigus et affectés par le transfert à 30%

0 . Ensuite, trois seuils séparant ces quatre

classes ont été désignés: Tl correspondant au transfert à 20%

0 (du jour 0 au jour 55); T2

correspondant aux stress aigus (du jour 56 à 70), et T3 correspondant au transfert à 30°/00

(du jour 71 à 105). A partir de ce modèle, on peut remarquer que sous le seuil Tl , 84% des

poissons n'ont pas été affectés par les infections opportunistes, alors qu'au-delà de ce seuil,

les infections apparaissent. Pour la famille R8, la majorité des poissons infectés est due

principalement au transfert à 20%

0 (ce qui représente 8.66%), ainsi qu 'au transfert à 30%

0

(6.66%), alors que seulement (0.66%) ont été affectés par les stress aigus. L'annexe 2

55

regroupe les données pour l'ensemble des familles. Il serait sans doute opportun de

considérer l'application d'un tel modèle pour des comparaisons statistiques ultérieures.

Afin de comparer les différences interfamiliales de résistance, l'approche du

« threshold/liability model» a été envisagée. Rappelons cependant que ce modèle ne doit

pas être vu comme une preuve scientifique, mais plus comme une hypothèse (Turnpenny et

Ellard, 2004). Dans les analyses à venir et ce avant la publication, l 'héritabilité sera

calculée. On pourrait envisager de donner un indice pour chaque individu en fonction du

seuil, lequel indice pourrait être utilisé comme valeur individuelle dans des calculs

d'héritabilité.

De plus, les croisements en demi-frère devraient être pris en compte. Il faudra

également regarder les différences de résistance en fonction du type d'hybrides. Les

données actuelles des facteurs de conditions indiquent que la majorité des familles sont en

bonne santé. Cependant, ces facteurs ont été calculés chez les poissons vivants. Il serait

donc intéressant de les comparer avec ceux des poissons morts (des mesures ayant été

prises pour chaque poisson mort) afin de vérifier si ce sont les plus petits qui étaient les

plus sensibles. Par contre les mortalités étant survenues à divers moments au cours de l'été,

il faudrait sans doute trouver le moyen de réajuster l'évolution des facteurs de condition en

fonction des degrés jours si on voulait procéder à des comparaisons.

56

CONCLUSION GÉNÉRALE

Ce travail a permis d'une part de dresser le répertoire des allèles du CMH de classe

II~ 1 des géniteurs. A partir du génotype parental, l'estimation des combinaisons alléliques

pouvant possiblement être retrouvées chez la progéniture a pu être établie. D'autre part, la

détermination des bases génétiques de la susceptibilité/résistance naturelle des ombles et

l'identification de l'agent pathogène opportuniste Flexibacter maritimus permettront de

cibler des individus naturellement plus résistants. Ces résultats serviront à la suite du projet

qui vise à déterminer si les différences de résistance sont corrélées à des différences

alléliques au niveau de ~ 1. La détermination du génotype de la progéniture confirmera

l' estimation allélique établie et permettra de mettre en évidence la présence ou l'absence

d'un allèle conférant une résistance accrue.

Ce travail s' insère dans un projet qui vise à mettre à la disposition de l'industrie les

outils génétiques de sélection de lignées performantes pour l' élevage d'ombles en milieu

côtier. On cherche ainsi à optimiser la production et améliorer l'adaptabilité à l'eau de mer

des populations d'élevage ciblées.

57

LISTE DES RÉFÉRENCES

Anacker, RL. , et Ordal, E.l 1959. Studies on the myxobacterium Chondrococcus

columnaris. 1. Serological typing. Journal ofBacteriology 78: 25-32.

Anderson, D.P. 1990. Immunological indicators: efTects ofenvironmental stress on immune

protection and disease outbreaks. American Fisheries Society Symposium 8: 38-50.

Avendafio-Herrera, R , Rodriguez, l, Magarifios, B., Romalde, lL. , et Toranzo, AE. 2004.

Intraspecific diversity of the marine fish pathogen Tenacibaculum maritimum as

determined by randomly amplified polymorphic DNA-PCR Journal of Applied

Microbiology 96: 871-877.

Avendafio-Herrera, R , Irgang, R, Nufiez, S., Romalde, lL., et Toranzo, AE. 2005 .

Recommendation of an appropriate medium for in vitro drug susceptibility testing of the

fish pathogen Tenacibaculum maritimum. Antimicrobial Agents and Chemotherapy 49: 82-

87.

Bensa, le. 2003 . Le complexe majeur d'histocompatibilité et la définition du SOl.

Transfusion Clinique et Biologie 10 : 209-213.

Bernadet, lF. 1997. Immunization with bacterial antigens: Flavobacterium and Flexibacter

infections. Developments in Biological Standardization 90: 179-188.

Bernatchez, L., et Landry, e. 2003 . MHC studies in nonmodel vertebrates: what have we

learned about natural selection in 15 years? Journal ofEvolutionary Biology 16: 363-377.

Birnboim, He. et Doly, l 1979. A rapid alkaline extraction procedure for screenmg

recombinant plasmid DNA Nucleic Acids Research 7: 1513-1519.

58

Bouillon, M., et Mourad, W.M. 2003 . Complexe majeur d'histocompatibilité de classe II :

diversité fonctionnelle. Medecine Sciences 19: 988-993 .

Budowle , B. Chakraborty, R , Giusti, AM., Eisenberg, Al, et Allen, Re. 1991. Analysis

of the VNTR locus DI S80 by the PCR followed by high-resolution PAGE. Arnerican

Journal ofHuman Genetics 48 : 137-144.

Cepeda, e. , Garcia-Marquez, S., et Santos, Y. 2003 . Detection of Flexibacter maritimus in

fish tissue using nested PCR amplification. Journal ofFish Diseases 26: 65-70.

Chevassus, B. , et Dorson, M. 1990. Genetics of resistance to disease in fishes. Aquaculture

85 : 83-107.

Cipriano, Re., Marchant, D., Jones, T.E., et Schachte, lH. 2002. Practical application of

disease resistance to furunculosis . Aquaculture 206: 1-17.

d'Autray Tarte, P. 2004. Analyse du CMH de classe IIp chez l'omble de fontaine

(Salvelinus fontinalis) et résistance à Aeromonas salmonicida. Mémoire présenté à la

faculté des études supérieures de l'Université de Montréal, département de Microbiologie et

Immunologie.

Difco TM et BBL TM Manual. 2003 . Manual of microbiological culture media. Difco

laboratories, Detroit. pp. 536-537.

Ellis, AE. 1981 . Stress and the modulation of defence mechanisms in fish . In: Stress and

fish (Pickering, AD. , ed.) . Academic Press, London and New York. pp. 209-245 .

Falconer, D.S. 1965. The inheritance of liability to certain diseases, estimated from the

incidence among relatives. Annals ofHuman Genetics 29: 51-76.

59

Falconer, D .S. et Mackay, F.C. 1996. Introduction to Quantitative Genetics. 4th Edition.

Addison Wesley Longman, New York. pp. 300-311.

Fevolden, S.E., Refstie, T. et Reed, KH. 1991. Selection for high and low cortisol stress

response in Atlantic salmon (Salmo safar) and rainbow trout (Oncorhynchus mykiss) .

Aquaculture 95 : 53-65.

Fevolden, S.E., Refstie, T., et Gjerde, B. 1993 . Genetic and phenotypic parameters for

cortisol and glucose stress response in Atlantic salmon and rainbow trout. Aquaculture 118:

205-216.

Fevolden, S.E ., Reed, KH., et Gjerde, B. 1994. Genetic components of post-stress cortisol

and lysozyme activity in Atlantic salmon; correlations to disease resistance. Fish and

Shellfish Immunology 4: 507-519.

Fevolden, S.E, Roed KH. , Fjalestad, KT., et Stien, J. 1999. Post-stress levels oflysozyme

and cortisol in adult rainbow trout (Oncorhynchus mykiss); heritabilities and genetic

correlations. Journal ofFish Biology 54: 900-910.

Fjalestad, KT., Gjedrem, T. , et Gjerbe, B. 1993 . Genetic improvement ofdisease resistance

in fish : an overview. Aquaculture 111 : 65-74.

Grimholt, u., Drables, F., Jorgensen, S.M., Hoyheim, B., et Stet, R.J.M. 2002. The major

histocompatibility class 1 locus in Atlantic salmon (Salmo salar L.): polymorphism, linkage

analysis and protein modelling. Immunogenetics 54: 570-581.

Grimholt, u., Getahun, A , Hermsen, T., et Stet, R. 2000. The major histocompatibility

class II alpha chain in salmonid fishes. Developmental and Comparative Immunology 24:

751-63 .

60

Grimholt, U , Larsen, S., Nordmo, R., Midtlyng, P., Kjoeglum, S., Storset, A , Saeb, S. , et

Stet, R.JM. 2003 . MHe polymorphism and disease resistance in Atlantic salmon (Salmo

salar); facing pathogens with single expressed major histocompatibility class 1 and class II

loci. Immunogenetics 55 : 210-219.

Henryon, M., Jokumsen, A , Berg, P., Lund, 1., Pedersen, P.B. , Olesen, N.J. , et

Slierendrecht, W.J 2002. Genetic variation for growth rate, feed conversion efficiency, and

disease resistance exists within a farmed population ofrainbow trout. Aquaculture 209: 59-

76.

Iwama, G., et Nakanishi, T. 1996. The specific immune system : cellular defenses. In:

Manning, M.J. , et Nakanishi, T. (ed.): The fish immune system organism, pathogen and

environment. Academic Press, London. pp. 185-205 .

Janeway, C.A 2001. How the immune system works to protect the host from infection: A

personal view. Proceedings of the National Academy of Sciences. 98: 7461-7468 .

Këllner, B. , Wasserrab, B. , Kotterba, G., et Fischer, U 2002. Evaluation of immune

functions of rainbow trout (Oncorhynchus mykiss) - how can environmental influences be

detected? Toxicology Letters 131: 83-95 .

Kumar, S., Tamura, K. , et Nei, M. 2004. MEGA 3 : Integrated software for molecular

evolutionary genetics analysis and sequence alignment. Briefings in Bioinformatics 5: 150-

163 .

Langefors, A , Lohm, J , Grahn, M., Andersen, O., et von Schantz, T. 2001. Association

between major histocompatibility complex class lIB alleles and resistance to Aeromonas

salmonicida in Atlantic salmon. Proceedings of the Royal Society of London Series B -

Biological Sciences 268: 479-485 .

61

Magarinos, B. , Pazos, F. , Santos, Y , Romalde, J.L. , et Toranzo, AE. 1995. Response of

Pasteurella piscicida and Flexibacter maritimus to the skin mucus of marine fish. Diseases

of Aquatic Organisms 21: 103-108.

Marsh S.G.E ., Parham, P., et Barber, L. D. 2000. The lll.,A factsbook. Academic Press, San

Diego. In: Stet, RlM., de Vries, B. , Mudde, K, Hermsen, T. , van Heerwaarden, l , Shum,

B.P., et Grimholt, U. 2002. Unique haplotypes of co-segregating major histocompatibility

class II A and class II B alleles in Atlantic salmon (Salma salar) give rise to diverse class II

genotypes. 2002. Immunogenetics 54: 320-331 .

Miller, KM., et Withler, RE. 1996. Sequence analysis of a polymorphic MHC class II

gene in Pacific salmon. Immunogenetics 43 : 337-351.

Morinville, G., et Rasmussen, lB. 2003 . Early juvenile bioenergetic differences between

anadromous and resident brook trout (Salvelinus fantinalis). Canadian Journal of Fisheries

and Aquatic Sciences 60: 401-410.

Nakanishi, T. , Aoyagi, K , Xia, c., Dijkstra, lM. , et Ototake, M. 1999. Specific cell-

mediated immunity in fish. Veterinary Immunology and Immunopathology 72: 101-109.

Ono, H , O'hUigin, c., Vincek, V., et Klein, l 1993 . Exon-intron organization of fish

major histocompatibility complex class II p genes. Immunogenetics 38: 223-234.

Pazos, F., Santos, Y , Maclas, AR, Nunez, S., et Toranzo, AE. 1996. Evaluation of media

for the successful culture of Flexibacter maritimus. Journal of Fish Diseases 19: 193-197.

Pickering, AD. 1992. Rainbow trout husbandry: management of the stress response.

Aquaculture 100: 125-139.

62

Pottinger, T.G., et Pickering, A.D. 1997. Genetic basis to the stress response : selective

breeding for stress-tolerant fish . In: Fish stress and health in aquaculture, Iwana, G.K,

Pickering, A.D., Sumpter, lP. , et Schreck, C.B. Cambridge University Press, Cambridge,

UK. pp. 171-193.

Reichenbach, H. 1999. The ecology of the myxobacteria. Environmental Microbiology 1:

15-21.

Roed, KH. , Fevolden, S.E. , et Fjalestad, KT. 2002. Disease resistance and immune

characteristics in rainbow trout (Oncorhynchus mykiss) selected for lysozyme activity.

Aquaculture 209: 91-101.

Sahoo, P.K , Meher, P.K, Mahapatra, KD., Saha, lN., Jana, RK, et Reddy, P. 2004.

Immune response in different fullsib families oflndian major carp, Labeo rohita, exhibiting

differential resistance to Aeromonas hydrophila infection. Aquaculture 238 : 115-125.

Santos, Y , Pazos, F., et Barja, lL. 1999. Flexibacter maritimus, causal agent of

flexibacteriosis in marine fish . pp. 1-6. In: G. Oliver (ed.), ICES identification leaflets for

diseases and parasites of fish and selfish, no. 55. International Council for the Exploitation

of the Sea, Copenhagen, Denmark.

Sokal, RR et Rohlf, FJ. 1995. Biometry. The principles and practice of statistics In

biological research. 3rd ed. W. H. Freeman & Co. , New York. p. 887.

Stet, RlM., de Vries, B., Mudde, K, Hermsen, T. , van Heerwaarden, l , Shum, B.P., et

Grimholt, U. 2002. Unique haplotypes of co-segregating major histocompatibility class II A

and class II B alleles in Atlantic salmon (Salmo salar) give rise to diverse class II

genotypes. 2002. Immunogenetics 54: 320-331 .

63

Sültmann, H., Mayer, W.E. , Figueroa, F., 0' hUigin, c., et Klein, 1. 1994. Organization of

MHC Class IIJ3 genes in the zebrafish (Branchydanio rerio). Genomics 23: 1-24.

Sunnucks, P ., Wilson, AC., Beheregaray, L.B., Zenger, K., French, 1., et Taylor, AC.

2000. SSCP is not so difficult: the application and utility of single-stranded conformation

polymorphism in evolutionary biology and molecular ecology. Molecular Ecology 9: 1699-

1710.

Suzuki, M., Nakagawa, Y., Harayama, S., et Yamamoto, S., 200l. Phylogenetic analysis

and taxonomic study of marine Cytophaga-like bacteria: proposaI for Tenacibaculum gen.

nov. with Tenacibaculum maritimum comb. nov. and Tenacibaculum ovolyticum comb.

nov., and description of Tenacibaculum mesophilum sp. nov. and Tenacibaculum

amylolyticum sp. nov. International Journal of Systematic and Evolutionary Microbiology

51: 1639- 1652.

Toranzo, AE., Magarifios, B., et Romalde, 1.L. 2005 . A review of the main bacterial fish

diseases in mariculture systems. Aquaculture 246: 37-6l.

Trevor, P.T.E. 1996. Infection and disease. In: Iwama, G., et T. Nakanishi. (ed.). The fish

immune system: Organism, Pathogen, and Environment. Academic Press, London. pp. 339-

366.

Turnpenny, P., et Ellard, S. 2004. Emery's elements ofmedical genetics. 1ih ed. Churchill

Livingstone. Chapter 9. Polygenic and multifactorial inheritance. pp. 137-146.

Vandeputte, M. , et Prunet, P. 2002. Génétique et adaptation chez les pOIsons:

domestication, résistance au stress et adaptation aux conditions de milieu. Institut National

de la Recherche Agronomique, productions animales 15 : 365-37l.

64

Van Muiswinkel, W.B., Wiegertjes, G.F. et Stet, RJ.M. 1999. The influence of

environmental and genetic factors on the disease resistance of fish. Aquaculture 172: 103-

110.

Wakabayashi, H., Hikida, et H., Masumura, K. 1986. Flexibacter maritimus sp. nov., a

pathogen of marine fishes . International Journal of Systematic Bacteriology 36: 396- 398.

Wendelaar Bonga, S.E. 1997. The stress response in fish. Physiological Reviews 77: 501-

625 .

Wiegertjes, G.F., Stet, RJ.M., Paramentier, H.K., et van Muiswinkel, W.B. 1996.

Immunogenetics of disease resistance in fish: a comparative approach. Developmental and

Comparative Immunology 20: 365-381.

Yang, RC., Dhir, N.K. et Yeh, F.C. 1998. Intraclass correlation of polychotomous

responses of lodgepole pine to infection of western gall rust: a simulation study. Silvea

Genetica 47: 108-115.

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ANNEXES

66

T1 T2 T3

0.66%

84% 8.66% 6.66%

Annexe 1. Exemple du "liability/threshold mode!" pour la famille résidente Salve lin us fontinalis R8 (se reporter au tableau 1).

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Grou2e Famille avant Tl de Tl à T2 de T2 à T3 T3 et 2lus Intraspécifique Al 95 .33% 3.33% 0% 1.33%

A2 9l.33% 8% 0% 0.66% A3 86.66% 13 .33% 0% 0% A4 100% 0% 0% 0% A6 100% 0% 0% 0%

AR1 92% 0% 2% 6% AR2 93 .33% 2% 0% 4.66% AR3 90.66% 0% 0% 9.33% AR4 98% 0.66% 0% 1.33% AR5 80.66% 0% 0.66% 18.66% R2 92% 6% 0% 2% R3 9l.33% 7.33% 0.66% 0.66% R6 96% 0% 0% 4% R7 100% 0% 0% 0% R8 84% 8.66% 0.66% 6.66%

Interspécifique Al 95.33% 3.33% 0% 1.33% A2 9l.33% 8% 0% 0.66% A3 86.66% 13 .33% 0% 0% A4 100% 0% 0% 0% A6 100% 0% 0% 0%

CF1 93 .33% 2% 0% 4.66% CF2 94% 0.66% l.33% 4% CF5 94% 2.66% l.33% 2% CF6 99.33% 0.66% 0% 0% CF7 100% 0% 0% 0% C2 98.66% 1.33% 0% 0% C4 76.66% 1.33% 2% 20% C5 96% 0.66% 0% 3.33% C6 94% 0.66% 0.66% 4.66% C9 93 .33% 2% 0% 4.66%

Annexe 2. Tableau présentant, pour les familles intraspécifiques Salvelinus fontinalis et interspécifiques Salvelinus alpinus - Salvelinus fontinalis, les proportions de populations pour chacune des quatre classes phénotypiques séparées par les trois seuils : avant Tl : pas de poissons infectés, de Tl à T2 : poissons susceptibles au passage en eau de mer à 20/00 ,

donc les plus sensibles aux infections opportunistes, de T2 à T3 : poissons un peu plus résistants mais sensibles à une surimposition de stress ai~us successifs, T3 et plus : poissons encore plus résistants mais sensibles au transfert à 30 / 00 .

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